Management of Diabetes and Hyperglycemia in Hospitalized Patients

ABSTRACT

Diabetes is the most prevalent metabolic disorder, and in 2021, the International Diabetes Federation estimated that it affected 537 million adults globally. In 2024, the United States Centers for Disease Control reported that 38.1 million adult Americans, or 14.7% of the adult population, have diabetes. Patients with diabetes have a 3-4-fold greater chance ofhospitalization compared to those without diabetes. In 2020, in the U.S., there were over 7.86 million hospital discharges for adults listed as having diabetes. Hyperglycemia, defined as a blood glucose greater than 140 mg/dl (7.8 mmol/l), isreported in 22-46% of non-critically ill hospitalized patients. Extensive data indicates that inpatient hyperglycemia, in patients with or without a prior diagnosis of diabetes, is associated with an increased risk of complications and mortality. In 2025, the American Diabetes Association (ADA) recommends that once therapy is initiated, a glycemic goal of 140–180 mg/dL (7.8–10.0 mmol/L) is recommended for most critically ill (ICU) individuals with hyperglycemia. More stringent individualized glycemic goals may be appropriate for selected critically ill individuals if they are achieved without significant hypoglycemia. However, for non-critically ill (non-ICU) individuals, a glycemic goal of 100-180 mg/dL (5.6-10.0 mmol/L) is recommended, if achieved without significant hypoglycemia. Insulin remains the best way to control hyperglycemia in the inpatient setting, especially in critically ill patients. Intravenously administered insulin is the preferredmethod to achieve the recommended glycemic target in the ICU. In 2025, the ADA changed its recommendations on using SGLT2 inhibitors in inpatients. They now suggest that in people with type 2 diabetes and heart failure, SGLT2 inhibitors may be started or continued if there are no contraindications (which include prolonged fasting or post-operative recovery). The use of GLP-1 receptor agonists was not recommended in previous guidelines because of the need for more safety and efficacy studies in the inpatient setting. However, increasing evidence indicates that treatment with oral agents such as DPP4 inhibitors, alone or combined with basal insulin, is safe and effective in general medicine andsurgery patients with mild to moderate hyperglycemia.

INTRODUCTION

Diabetes is the most prevalent metabolic disorder, affecting more than 537 million adults globally and is projected to rise to almost 800 million (10.9% of the adult population) by 2045 (1). In the United States, data from the National Diabetes Statistics Report in 2023 estimated that 38.4 million people of all ages or 14.7% of all U.S. adults had diabetes (2). The percentage of the population with diagnosed diabetes is expected to rise, with one study projecting that as many as onein three U.S. adults will have diabetes during their lifetime (3). People with diabetes have a 35% greater chance of referral for elective operations and a 3-4-fold greater chance of hospitalization compared to those without diabetes (4-7). Data from the US and Scotland estimate that of those individuals with a discharge diagnosis of diabetes, 30% will require two or more hospitalizations in any given year (5; 6; 8). In 2020, in the U.S., there were over 7.86 million hospital discharges for adults listed as having diabetes, (i.e., diabetes as either a principal diagnosis for hospitalization or as a secondary diagnosis, coexisting condition) (9). Data from the USA suggest that the prevalence of diabetes in the adult inpatient population has increased by 2.5% annually from 17.1% to 27.3% between 2000 and 2018 (10). In the UK, the annual National Diabetes Inpatient Audit suggested that the prevalence of diabetes amongst inpatients had risen from15% in 2010 to almost 20% in 2019 (11). In addition, those hospitalized with a diagnosis of diabetes stay in the hospitalfor longer than those without a diagnosis of diabetes admitted for the same condition (12; 13).

Diabetes was the 8^th leading cause of death in the United States in 2021, accounting for 31.1 deaths per 100,000 of thepopulation (2). A further 120.3 per 100,000 people had diabetes listed as a contributing factor towards the cause of death (2). Not only does diabetes have a significant economic impact on those living with the condition, but it also imposes a substantial burden on the economy, with a total estimated cost of treating people diagnosed with diabetes in the UnitedStates in 2022 of $413 billion – or 25% of all health care spending in the US (14). This included $306.6 billion in directmedical costs. It is estimated that a further cost of $96.5 billion is incurred due to reduced productivity (14). Data from Ireland estimated that the overall cost of treating diabetes represented between 12 and 14% of the annual health budget. The cost per admission for someone with type 1 or type 2 diabetes was €4,027 and €5,026, respectively (15). Globally,diabetes care costs have been estimated at $1.3 trillion, rising to an estimated $2.1-2.5 trillion by 2030 (16; 17). This represents a rise in spending on diabetes as a proportion of global gross domestic product from 1.8% in 2015 to 2.2% in 2030 (17). Other than the costs of diabetes medications, the most significant component of this medical expenditure is hospital inpatient care (13; 18).

Hyperglycemia is defined as a blood glucose concentration greater than 140 mg/dl (7.8 mmol/l) (19-21). It is not just found in those with a pre-existing diagnosis of diabetes but in those with stress hyperglycemia or previously undiagnosed diabetes. The prevalence has been reported to be 22% to 46% in non-critically ill hospitalized patients (8; 19). Extensiveobservational and trial data indicate that inpatient hyperglycemia, in patients with or without a prior diagnosis of diabetes,is associated with an increased risk of complications and mortality, a longer hospital stay, a higher admission rate to the intensive care unit (ICU), and a higher need for transitional or nursing home care after hospital discharge (8; 22; 23).

Several studies and meta-analyses have shown that attempting ‘tight’ glycemic control using intensive insulin therapy isassociated with an increased risk of hypoglycemia (24-28), which has been associated with increased morbidity andmortality in hospitalized patients (19; 29-34). Thus, while insulin therapy is recommended for managing hyperglycemia inhospitalized patients, the concern about hypoglycemia has led leading professional organizations worldwide to recommend targets that avoid the risk of hypoglycemia (20; 27; 35-38).

This chapter reviews the pathophysiology of hyperglycemia during illness, the mechanisms for increased complicationsand mortality due to hyperglycemia and hypoglycemia, and the evidence supporting different therapies and approaches for the management of inpatient diabetes and hyperglycemia in critical care, general medicine, and surgical settings.

PREVALENCE OF DIABETES AND HYPERGLYCEMIA IN THE HOSPITALIZED PATIENT

Observational studies have reported a prevalence of hyperglycemia and diabetes ranging from 38% to 40% in hospitalized patients (8) and in 70-80% of those with diabetes who have a critical illness or cardiac surgery (39-41). A2017 report using point-of-care bedside glucose tests data in almost 3.5 million people (653,359 ICU and 2,831,436 non-ICU) from 575 hospitals in the United States reported a prevalence of hyperglycemia (defined as a glucose level >180mg/dl [10.0 mmol/l]) of 32.2% in ICU patients and in 32.0% of non-ICU patients (39). A study of 893 people across 69 ICUs in France reported a prevalence of hyperglycemia (>180 mg/dl [10 mmol/l]) of 45% (42). Other USA data suggest that between 2000 and 2018, the prevalence of diabetes amongst adult inpatients increased by 2.5% per year from 17.1% to 27.3% (10), and that over 33% of all hospital discharges in 2020 had diabetes listed as a diagnosis (9). However, this does not include those individuals who develop stress hyperglycemia. The American Diabetes Association (ADA) and American Association of Clinical Endocrinologists (AACE) consensus on inpatient hyperglycemia defined stress hyperglycemia or hospital-related hyperglycemia as any blood glucose concentration >140 mg/dl (>7.8 mmol/l) in patients without a prior history of diabetes (19; 20). The data from the US included those with newly identified diabetes or stress hyperglycemia as well as those with a prior diagnosis of diabetes (39). Although stress hyperglycemia typicallyresolves as the acute illness or surgical stress abates, a significant proportion (up to 60% in some reports) develop confirmed diabetes at 6-12 months after discharge (43; 44). A guide from the UK on the management of ‘diabetes at the front door’, also recommends that any individual without diabetes who presents acutely unwell should have a capillary glucose measurement and blood/urine ketone measurement taken, but that if it is high on admission (i.e. >140mg/dl [7.8 mmol/l]) and subsequently goes down to normal, then a diagnosis of stress hyperglycemia should be made and documented to the primary care team (21).

Measurement of HbA_1c is indicated in people with hyperglycemia without a history of diabetes to differentiate betweenstress-induced hyperglycemia and previously undiagnosed diabetes (21; 45-48). The ADA also recommends that an HbA_1c be done in those with diabetes who have not had it measured in the preceding 3 months (48). The Endocrine Society and the UK Joint British Diabetes Societies for Inpatient Care (JBDS) recommendations indicate that people hospitalized with elevated blood glucose >140 mg/dl (7.8 mmol/l) and an HbA_1c of 6.5% (48 mmol/mol) or higher can be identified as having diabetes (19; 21). Given the increasing prevalence of diabetes, the UK has also produced a calculator to help teams work out their optimal staffing levels (49).

PATHOPHYSIOLOGY OF HYPERGLYCEMIA DURING ILLNESS

In subjects without diabetes during the fasted state, plasma glucose is maintained between 70 – 100 mg/dl (3.9 – 5.6 mmol/l) by a finely regulated balance between glucose production from the liver and kidneys and glucose utilization inperipheral tissues. Maintenance of near-normal glucose concentration is essential for cardiovascular and central nervous system function because the brain can neither synthesize nor store glucose (50; 51).

Systemic glucose balance is maintained by dynamic, minute-to-minute regulation of endogenous glucose production and glucose utilization by peripheral tissues (52). Glucose production is accomplished by gluconeogenesis or glycogenolysisprimarily in the liver and, to a lesser degree, by the kidneys (53; 54). Gluconeogenesis results from converting non-carbohydrate precursors such as lactate, alanine, and glycerol to glucose in the liver (55). Excess glucose is polymerized into glycogen, mainly stored in the liver and muscle. Hyperglycemia develops because of three processes: 1) increased gluconeogenesis, 2) accelerated glycogenolysis, and 3) impaired glucose utilization by peripheral tissues (Figure 1).

Figure 1. Pathogenesis of hyperglycemia. Hyperglycemia results from increased hepatic glucose production and impaired glucose utilization in peripheral tissues. Reduced insulin and excess counter-regulatory hormones (glucagon, cortisol, catecholamines, and growth hormone) increase lipolysis and protein breakdown (proteolysis) and impair glucose uptake by peripheral tissues. Hyperglycemia causes osmotic diuresis, leading to volume depletion, decreasing glomerular filtration rate, and worsening hyperglycemia. At the cellular level, increased blood glucose concentrations result in mitochondrial injury by generating reactive oxygen species and endothelial dysfunction by inhibiting nitric oxide production. Hyperglycemia increases levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin [IL]-6, leading to immune system dysfunction. These changes can eventually lead to an increased risk of infection, impaired wound healing, multiple organ failure, prolonged hospital stay, and death. Adapted from ref (25).

From the quantitative standpoint, inappropriately increased hepatic glucose production represents the major pathogenic disturbance. Increased hepatic glucose production results from the high availability of gluconeogenic precursors. These include the amino acids alanine and glutamine, which result from accelerated proteolysis and decreased proteinsynthesis; lactate, which results from increased muscle glycogenolysis; glycerol, which results from increased lipolysis;and the increased activity of gluconeogenic enzymes (phosphoenol pyruvate carboxykinase, fructose-1,6-bisphosphatase, and pyruvate carboxylase) (53; 55).

Glucose metabolism is maintained by an interaction of glucoregulatory hormones – insulin and counter-regulatory hormones (glucagon, cortisol, epinephrine, norepinephrine, and growth hormone). Insulin controls hepatic glucose production by suppressing hepatic gluconeogenesis and glycogenolysis. Depending on the concentration in the circulation, insulin inhibits glycogenolysis and protein breakdown and, at higher concentrations, promotes protein anabolism ininsulin-sensitive tissues such as muscle, glucose uptake, and glycogen synthesis (52; 56; 57). In addition, insulin is a potent inhibitor of lipolysis, free fatty acid oxidation, and ketogenesis (56-58).

Counter-regulatory hormones also play an essential role in regulating glucose production and utilization. Glucagon is themost important glycogenolytic hormone, and therefore regulates hepatic glucose production in healthy individuals and in every state of hyperglycemia (53). During stress, excess concentration of counter-regulatory hormones results in altered carbohydrate metabolism by inducing insulin resistance, increasing hepatic glucose production, and reducing peripheralglucose utilization. In addition, high epinephrine levels stimulate glucagon secretion and inhibit insulin release bypancreatic β-cells (59; 60).

The development of hyperglycemia results in an inflammatory state characterized by an elevation of pro-inflammatory cytokines and increased oxidative stress markers (61-63). Circulating levels of TNF-α, IL-6, IL1-ß, IL-8, and C-reactiveprotein are significantly increased two- to fourfold on admission in people with severe hyperglycemia compared withcontrol subjects, and levels returned to normal levels after insulin treatment and resolution of hyperglycemic crises (61).Raised concentrations of TNF-α lead to insulin resistance at the level of the insulin receptor and through altered regulationof the insulin-signaling pathway (62; 64). In addition, preventing insulin-mediated activation of phosphatidylinositol 3-kinase TNF-α reduces insulin-stimulated glucose uptake in peripheral tissues (62; 64; 65).

CONSEQUENCES OF HYPERGLYCEMIA IN THE HOSPITALIZED PATIENTS

A large body of literature, including observational and prospective randomized clinical trials, in people with and withoutdiabetes, as well as those who are critically or non-critically ill has shown a strong association between hyperglycemia (in particular, a blood glucose >200mg/dl [11.0mmol/l]) and poor clinical outcomes, such as mortality, infections, and hospital complications compared to those with a glucose concentration of <100mg/dl (5.6mmol/l) (5; 66-76). This association correlates with the severity of hyperglycemia prior to or on admission and during the hospital stay (72; 77-79). Of interest, increasing evidence indicates an increased risk of complications and mortality in patients without a history of diabetes(stress-induced) compared to patients with a known diagnosis of diabetes (8; 69; 75; 77; 80; 81). It is not clear if stresshyperglycemia is the direct cause of poor outcomes or if it is a general marker of the severity of illness. However, there are data to show that those without a prior history of diabetes have fewer point-of-care glucose concentrations measured compared to those with diabetes, even when glucose concentrations are just as high (75; 82). In those who had diabetes, having more point-of-care tests increases contact with the ward staff, suggesting that impending complications may be picked up sooner, resulting in lower mortality. These data correlate with other work that also shows that those with lower preoperative HbA_1c lower the number of post-operative glucose checks in a general surgical population (83).

The mechanisms implicated in the detrimental effects of hyperglycemia during acute illnesses are not entirely understood.Current evidence indicates that severe hyperglycemia results in impaired neutrophil granulocyte function, high circulating free fatty acids, and overproduction of pro-inflammatory cytokines and reactive oxygen species (ROS) that can result in direct cellular damage and endothelial and immune dysfunction (84; 85).

The majority of evidence linking hyperglycemia and poor outcomes comes from studies in the ICU. Falciglia et al., in a retrospective study of over 250,000 veterans admitted to various ICUs, reported that hyperglycemia is an independentrisk factor for mortality and complications (77). In a nonrandomized, prospective study, Furnary et al. followed 3,554 people with diabetes who underwent coronary artery bypass graft. These were treated with either intermittent subcutaneous insulin (SCI) or with a continuous intravenous insulin infusion (CIII). The group treated with SCI achieved an average blood glucose of 214 mg/dl (11.9 mmol/l), compared to 177 mg/dl (9.8 mmol/l) in the CIII group. The CIII group had significantly fewer deep sternal wound infections and a 50% lower risk-adjusted mortality (73; 86). In other ICU studies, patients with blood glucose levels >200 mg/dl (>11.1 mmol/l) were shown to have higher mortality comparedto those with blood glucose levels <200 mg/dl (<11.1 mmol/) (72; 75). Importantly however, once again it has been shown that it was those people who were not previously known to have diabetes yet who developed hyperglycemia on the ICU who fared worse (75; 87). This was confirmed by another ICU study looking at almost 350,000 people, looking at the outcomes of those with sepsis (88). These authors showed that having hyperglycemia without a prior diagnosis of diabetes was associated with an increased stay in hospital and ICU and greater 90-day mortality (88). However, there was no difference in outcomes for those with diabetes unless they had experienced severe hypoglycemia (<40 mg/dl [2.2 mmol/l]), in which case mortality rose (OR 2.95 95%CI 1.19-7.32) (88). Another ICU study randomized 9230 people who were not given early parenteral nutrition to liberal glucose control (insulin only started if glucose rose to >215 mg/dl [>11.9 mmol/l]), or tight glucose control with glucose concentrations maintained between 80 and 110 mg/dl (4.4 – 6.1 mmol/l). These authors showed no differences in outcome, including length of time in ICU, infection rates, time on respiratory or hemodynamic support, or mortality. The only differences were lower severe acute kidney injury incidence and cholestatic liver dysfunction in the tight glycemic control arm (89).

The association of hyperglycemia and poor outcomes also applies to those not in ICU but admitted to general medicine, surgery, or mental health services. In such individuals, hyperglycemia is associated with poor hospital outcomes, including prolonged hospital stay, infections, disability after hospital discharge, and death (5; 8; 66; 67; 81; 90). In a study of 1,886 patients admitted to a community hospital, mortality in the general floors was significantly higher in patients withnewly diagnosed hyperglycemia and with known diabetes compared to subjects with normal glucose values (10% vs. 1.7% vs. 0.8%, respectively, p < 0.01) (8). In a prospective cohort multicenter study of 2,471 patients with community-acquired pneumonia, those with an admission glucose level of >198 mg/dl (>11.0 mmol/l) had a greater risk of mortality and complications than those with glucose <198 mg/dl (<11.0 mmol/l) (91). The risk of complications increased by 3% foreach 18 mg/dl (1.0 mmol/l) increase in admission glucose (91). In a retrospective study of 348 patients with chronic obstructive pulmonary disease and respiratory tract infection, the relative risk of death was 2.1 in those with a bloodglucose of 126-160 mg/dl (7.0-8.9 mmol/l), and 3.4 for those with a blood glucose of >162 mg/dl (9.0 mmol/l) compared topatients with a blood glucose of 108 mg/dl (6.0 mmol/l) (92). Similar data from a systematic review and meta-analysis from 38 studies of people who needed hospitalization for community-acquired pneumonia showed that in those without a prior diagnosis of diabetes, hyperglycemia was associated with an almost doubling of the need for ICU admission (crude OR 1.82, 95% CI 1.17 to 2.84) and in-hospital mortality (adjusted OR 1.28, 95% CI 1.09 to 1.50) (81). Those people already known to have diabetes had no increased risk of either.

General surgery patients with hyperglycemia during the perioperative period are also at increased risk for adverse outcomes. Reviews of diabetes and the risk of surgical site infection across a variety of surgical specialties have shown that high peri-operative glucose is associated with an increased risk of infection (93; 94). In a case-control study, elevated preoperative glucose levels increased the risk of postoperative mortality in patients undergoing elective non-cardiac non-vascular surgery (95). Patients with glucose levels of 110-200 mg/dl (5.6-11.1 mmol/l) and those with glucose levels of >200 mg/dl (>11.1 mmol/l) had, respectively, 1.7-fold and 2.1-fold increased mortality compared to those with glucose levels <5.6 mmol/l (<110 mg/dl) (95). In another study, patients with glucose levels >220 mg/dl (>12.2 mmol/l) on the first postoperative day had a rate of infection 2.7 times higher than those who had serum glucose levels <220 mg/dl (<12.2 mmol/l) (96). Other authors showed an increase of postoperative infection rate by 30% for every 40mg/dl (2.2 mmol/l) rise in postoperative glucose level above 110 mg/dl (6.1 mmol/l) (96). Further, a study looking at perioperativeglycemic control and the effect on surgical site infections in people with diabetes undergoing foot and ankle surgery showed that 11.9% of those with a serum glucose ≥200 mg/dl (11.1 mmol/l) during the admission developed a surgicalsite infection versus only 5.2% of those with a serum glucose <200 mg/dl (11.1 mmol/l) (odds ratio = 2.45; 95% CI 1.09-5.52, P = 0.03) (97). Lastly, a prospective randomized study looking at the impact of glycemic control at 1-year post livertransplant showed that in those randomized to glycemic control of blood glucose below 140 mg/dl (7.8 mmol/l), any infection within one year occurred in 35 of the 82 patients (42.7%) versus 54 of 82 (65.9%) in those randomized to glycemic control of 180 mg/dl (10.0 mmol/l) (P = 0.0046) (98).

Emerging evidence suggests that early intervention and the use of technology allowing proactive identification of people at risk help to reduce hospital-acquired infection rates, episodes of hyper- and hypoglycemia, and, in some cases, length of stay (99-102). A meta-analysis also shows that improving peri-operative glycemic control reduced postoperative infection rates (103).

In summary, despite a large amount of work having been done, and the numerous data showing the association – but not causation – between hyperglycemia and poor outcomes, and because there remain a very few robust intervention studies showing a benefit of glycemic control, the optimal blood glucose concentration for people on ICU has yet to be determined (104; 105).

GLYCEMIC TARGETS IN THE ICU AND NON-ICU SETTINGS

The American Diabetes Association (ADA) and American Association of Clinical Endocrinology (AACE) task force on inpatient glycemic control and other groups recommended differing glycemic targets in the ICU setting (20) (Table 1). These guidelines suggest targeting a BG level between 140 and 180 mg/dl (7.8 and 10.0 mmol/l) for the majority of ICU patients and a lower glucose target between 110 and 140 mg/dl (6.1 and 7.8 mmol/l) in selected ICU patients (i.e., centers with extensive experience and appropriate nursing support, cardiac surgical patients, patients with stableglycemic control without hypoglycemia). Glucose targets >180 mg/dl (>10.0 mmol/l) or <110 mg/dl (<6.1 mmol/l) are not recommended in ICU patients. There is an argument that lowering glucose thresholds for hospital patients will likely be associated with harm (32). Still, an equally persuasive argument suggests that implementing the thresholds advocated by national and organizational guidelines has led to safer care (106).

The Society of Critical Care Medicine (SCCM) guidelines for the management of hyperglycemia in critically ill (ICU)patients recently “recommended against” titrating an insulin infusion to a lower glucose target of 80–139 mg/dL (4.4–7.7 mmol/L) as compared with a higher BG target range of 140–200 mg/dL (7.8–11.1 mmol/L) to reduce the risk of hypoglycemia (107). They also recommended that clinicians should initiate glycemic management protocols and procedures to treat persistent hyperglycemia greater than or equal to 180 mg/dL (10 mmol/L) to maintain target glucose below <180 mg/dl (<10.0 mmol/l) in critically ill adults (107). They also suggest that the insulin regimen and monitoringsystem be designed to avoid and detect hypoglycemia (blood glucose <70 mg/dl [<3.9 mmol/l]) and to minimize glycemicvariability.

Table 1. Major Guidelines for Treatment of Hyperglycemia in a Hospital Setting
	ICU	Non-ICU
ADA/AACE (20; 108)	Initiate insulin therapy for persistent hyperglycemia (glucose >180 mg/dl [>10 mmol/l]).Treatment goal: For most people, target a glucose level between 140 – 180 mg/dl (7.8 – 10.0 mmol/l].More stringent goals (110 – 140 mg/dl [6.1 – 7.8 mmol/l]) or 100 – 180 mg/dL (5.6 –10.0 mmol/L), may be appropriate for selected patients and are acceptable if they can be achieved without significant hypoglycemia	No specific guidelines.Insulin therapy should be initiated for the treatment of persistent hyperglycemia ≥180 mg/dL (10.0 mmol/L) and targeted to a glucose range of 140 –180 mg/dL (7.8 – 10.0 mmol/L) for most critically ill patients. More stringent goals, such as 110–140 mg/dL (6.1–7.8 mmol/L), may be appropriate for selected patients (e.g., critically ill postsurgical patients or patients with cardiac surgery) as long as they can be achieved without significant hypoglycemia.Less stringent targets (e.g., >250 mg/dL (13.9 mmol/L) maybe appropriate in people withsevere comorbidities or end of life care.
ACP (27)	Recommends against intensiveinsulin therapy in those with orwithout diabetes in surgical / medical ICUs Treatment goal: target glucosebetween 140 – 200 mg/dl (7.8 – 11.0 mmol/l), in people with or without diabetes, in surgical / medical ICUs
Critical Care Society (107)	BG >180 mg/dl (>10.0 mmol/l) should trigger insulin therapy. Treatment goal: maintainglucose <180 mg/dl (<10.0 mmol/l) for most adults in ICU. Maintain glucose levels <180mg/dl (10.0 mmol/l) while avoiding hypoglycemia.
Endocrine Society (19; 109)		Pre-meal glucose target <140mg/dl (<7.8mmol/l) and random blood glucose <180 mg/dl (<10.0 mmol/l). Those with insulin treated diabetes aim for a target glucose of 100 – 180 mg/dL (5.6 – 10 mmol/L). A lower target range may beappropriate in people able toachieve and maintain glycemiccontrol without hypoglycemia. Aglucose of <180 – 200 mg/dl(<10.0 – 11.0 mmol/l) isappropriate in those withterminal illness and/or withlimited life expectancy or at high risk for hypoglycemia. Adjust antidiabetic therapywhen glucose falls <100 mg/dl (<5.6 mmol/l) to avoidhypoglycemia.
Society of ThoracicSurgeons (110)	Continuous insulin infusionpreferred over SC or intermittent intravenousboluses. Treatment goal: Recommendglucose <180 mg/dl (<10.0 mmol/l) during surgery (≤110 mg/dl [≤6.1 mmol/l] in fasting and pre-meal states)
Joint British Diabetes Society for Inpatient Care(111)		Target blood glucose levels inmost people of between 108 – 180 mg/dl (6.0 – 10 mmol/l) with an acceptable range of between 72 – 216 mg/dl (4.0 – 12.0 mmol/l).

AACE/ADA, American Association of Endocrinologists and American Diabetes Association joint guidelines; ACP, American College of Physicians; ADA, American Diabetes Association; ICU, intensive care unit; SC, subcutaneous.

In the non-ICU setting, the Endocrine Society and the ADA/AACE Practice Guidelines recommended a pre-meal glucose of <140 mg/dl (<7.8 mmol/l) and a random BG of <180 mg/dl (<10.0 mmol/l) for the majority of non-critically ill patients treated with insulin (19; 20; 35; 109). More recently, the American Diabetes Association has recommended that targetglucose for most general medicine and surgery patients in non-ICU settings should be between 140 – 180 mg/dl (7.8 – 10.0 mmol/l) (108). In contrast, higher glucose ranges (>200 mg/dl [>11.1 mmol/l]) may be acceptable in people who are terminally ill or in those with severe comorbidities as a way of avoiding symptomatic hyperglycemia (19; 112).

Guidelines from the JBDS in the UK published over the last few years aim for target blood glucose concentrations in most people between 108 – 180 mg/dl (6.0 – 10.0 mmol/l) with an acceptable range of between 72 – 216 mg/dl (4.0 –12.0 mmol/l) (111). Table 1 summarizes the currently available guidelines for managing hyperglycemia in the hospitalsetting.

EVIDENCE FOR CONTROLLING HYPERGLYCEMIA IN ICU AND NON – ICU SETTINGS

In 2001, the Leuven surgical ICU study promoted intensive glycemic control in the critical care setting (113). This study randomized 1,548 people admitted to the surgical ICU (63% cardiac cases, 13% with diabetes, with most receiving earlyparenteral nutrition). Individuals were randomized to either conventional therapy, with target glucose between 180 – 200 mg/dl (10.0 – 11.1 mmol/l), or intensive treatment to target glucose between 80 – 110 mg/dl (4.4 – 6.1 mmol/l). Those in the conventional arm had a mean daily glucose average of 153 mg/dl (8.5 mmol/l), and those in the intensive arm had an average glucose of 103 mg/dl (5.7 mmol/l). Those in the intensive group had significantly less bacteremia, fewer antibioticrequirements, lower length of ventilator dependency, lower number of ICU days, and an overall 34% reduction in mortality(113). Following a similar study design, the same group of investigators randomized people to a medical ICU (18% withdiabetes) and reported that intensive insulin therapy (mean daily glucose of 111 mg/dl [6.2 mmol/l]) resulted in less ICU and total hospital complications in those with three days of insulin treatment (114). These two studies together, based on the positive outcomes on morbidity and mortality, suggested a glycemic target in the ICU of 140 – 180 mg/dl (7.8 – 10.0mmol/l) (20). There was also a realization that while lower targets may be appropriate for selected individuals, a target of <110 mg/dl (<6.1 mmol/l) was not recommended (20).

Many well-designed randomized controlled trials and meta-analyses have shown that such low glucose targets aredifficult to achieve, even in environments with high staff-to-patient ratios, without increasing the risk for severe hypoglycemia (24; 115-117). In addition, these and other studies failed to show improvement in clinical outcomes andhave even shown increased mortality risk with intensive glycemic control, targeting glucose concentrations of (80 – 110 mg/dl [4.4 – 6.1 mmol/l]) versus conventional glycemic control (140 – 200 mg/dl [7.8 – 11.0 mmol/l]) (Table 2) (29; 115-118). Most of these studies showed no differences in clinical outcomes between groups but had an increased risk of severe hypoglycemia in the intensively treated arms. One study in ICU patients was the Normoglycemia in Intensive Care Evaluation and Surviving Using Glucose Algorithm Regulation (NICE-SUGAR) trial, which randomized over 6104subjects to receive either conventional glycemic control to target glucose <180 mg/dl [<10.0 mmol/l]) or intensiveglycemic control (target 81 – 108 mg/dl [4.5 – 6.0 mmol/l]). This study also reported no difference in hospital mortality butfound increased mortality at 90 days of follow-up (24.9% vs. 27.5%, p=0.02) (24). In a subsequent analysis of the trial, the NICE-SUGAR investigators reported a higher frequency of hypoglycemia in the intensive arm (6.8% vs. 0.5%), and those with hypoglycemia had a ~2-fold increase in mortality compared to patients without hypoglycemia (29). More recently, Gunst et al. recently published the results of a multicenter, randomized trial involving 9230 patients in medical and surgical ICUs (89). 4622 patients were assigned to liberal glucose control, where insulin was initiated only when the blood glucose level was above 215 mg per deciliter (11.9 mmol per liter), and 4608 patients were assigned to tight glucose control, targeting a glucose level between 80 and 110 mg per deciliter. The primary outcome, the duration of time in ICU care, did not differ significantly between the two trial groups. The hazard ratio for earlier discharge alive with tight glucose control was 1.00 with a 95% confidence interval of 0.96 to 1.04. Effective glycemic separation between the groups was observed, with a median absolute difference of -28 mg/dl (-1.6 mmol/l) in daily blood glucose levels. Additionally, the safety outcome, mortality within 90 days after randomization, was 10.1% in the liberal-control group and 10.5% in the tight-control group. The incidence of other secondary outcomes, including severe hypoglycemia, time to discharge alive from the hospital, use of respiratory support, or in-hospital mortality, were no different between intensive and relaxed glycemic targets, except for a trend in lower rates of liver and kidney injury in the tight control group. Umpierrez recently summarized the data on mortality and outcomes of ICU RCTs (119).

Increasing evidence indicates that high pre-admission glycemic control – as measured by HbA_1c >8.0% (64mmol/mol) is associated with lower mortality than those with an HbA_1c <6.5% (48mmol/mol) (120). Whether this is due to an increased risk of hypoglycemia in the low HbA_1c group or an increased frequency of monitoring or bedside vigilance in those with higher glucose or preadmission HbA_1c remains unknown (75; 83).

Table 2. Clinical Trials of Intensive Glycemic Control in ICU Populations
Study	Setting	Population	Percentage with diabetes	Clinical Outcome
Malmberg, 1994 (121)	CCU	People with diabetes with suspected or confirmed acute MI	100	28% decreasemortality after 1 year
Furnary, 1999 (73)	CCU	People with diabetes undergoing CABG	100	65% decrease indeep sternal woundinfection rate
Van den Berghe,2001 (113)	Surgical ICU	Mixed, with CABG	13	34% decrease in mortality
Furnary, 2003 (86)	CCU	People with diabetes undergoing CABG	100	50% decrease inadjusted mortalityrate
Krinsley, 2003 (72)	Medical and surgical ICU	Mixed	22.4	27% decrease inmortality
Lazar, 2004 (122)	Operating room and ICU	People with diabetes undergoing CABG	100	60% decrease of post - operative atrialfibrillation
Van den Berghe,2006 (114)	Medical ICU	Mixed	17	18% decreasemortality
Gandhi, 2007 (123)	OperatingRoom	Mixed, undergoing cardiac surgery	19.6	No difference inmortality; increase instroke rate in the intensive treatmentarm
VISEP, 2008 (115)	Medical ICU	Mixed, admitted withsepsis	30	No differences in 28-day or 90-daymortality, end-organ failure, length of stay
De La Rosa, 2008(116)	Medical and surgical ICU	Mixed	12	No differences in 28-day mortality orinfection rate
Glucontrol, 2009 (124)	Medical and surgical ICU	Mixed	18	No difference in 28-day mortality
NICE-SUGAR,2009/2012 (24; 29)	Medical and surgical ICU	Mixed	20	No difference in 90-day mortality
Boston Children’s(SPECS), 2012 (125; 126)	Cardiac ICU	Cardiac surgery,people without diabetes	0	No differences in 30-day mortality, length of stay, in the cardiac ICU, length ofhospital, duration ofmechanicalventilation andvasoactive support,or measures of organfailure
ChiP, 2014 (127)	Pediatric ICU	Criticalillness/injury/majorsurgery, those without diabetes.	0	No difference in 30-day mortality.Increasedhypoglycemia in theintensive treatedgroup
CGAO–REA, 2014 (128; 129)	Medical ICU	Mixed	23	No difference in 90-day mortality. Increasedhypoglycemia in theintensive treatedgroup
Okabayashi, 2014 (130)	Surgical ICU	Mixed	25.3	Decreased surgicalsite infection in theintensive treatedgroup
Umpierrez (GLUCOCABG) 2015	Surgical ICU	CABG	50%	No difference in mortality
Gunst et al ICU	ICU		XX	No difference in mortality

MI, myocardial infarction, ICU – Intensive Care Unit, CABG – Coronary artery bypass graft. Mixed-study enrolled those with and without diabetes.

The GLUCO-CABG trial was a randomized open-label clinical study that included those with and without diabetesundergoing CABG who experienced perioperative hyperglycemia, defined as a BG >140 mg/dl (>7.8 mmol/l) 6069 (70). A total of 302 people between 18 and 80 years of age were randomized to the intensive glycemic control group (target BG 100 – 140 mg/dl [5.6 – 7.8 mmol/l]) or the control group (BG 141 – 180 mg/dl [7.9 – 10.0 mmol/l]) in the ICU. Aftertransitioning from the ICU to the telemetry floor, patients were managed with a single treatment protocol to maintain a glucose target of <140 mg/dl (<7.8 mmol/l) before meals during the hospital stay. The primary outcome included differences between intensive and conservative glucose control on a composite of perioperative complications, includingsternal wound infection, bacteremia, respiratory failure, pneumonia, acute kidney injury, major adverse cardiovascular events including acute coronary syndrome, stroke, heart failure, and cardiac arrhythmias (70). The mean BG during the ICU stay was 132±14 mg/dl (7.3±0.8 mmol/l) in the intensive and 152±17 mg/dl (8.4±1.0 mmol/l) in the conservative group. Intensive glucose treatment resulted in a 20% reduction in perioperative complications compared to theconservative group (42% vs. 52%). Of interest, there were no differences in the rate of complications among patients with diabetes treated with intensive or conservative regimens (42% vs. 52%, p=0.08); however, intensive treatment wasassociated with a significantly lower rate of complications compared to the conservative group in those without diabetes (34% vs. 55%, p=0.008) (70). Hospitalization costs were lower in the intensive group (median [IQR] $36,681 [28,488 – 46,074] vs. $40,913 [31,464 – 56,629], p=0.04), with an average total cost savings of $3,654 per case compared to conservative glucose control (131).

To date, few large studies have been conducted to determine if improved control in those not in ICU may result inreduced morbidity and mortality in general medical and surgical patients – indeed, until recently, for most people in hospital with diabetes while there are observational data to show that dysglycemia is harmful, there were little data to show that improving glycemic control helps (132). A randomized controlled trial from 2011 reported that improved glucose control using a basal-bolus regimen may reduce hospital complications in general surgery patients (71). Improving glucose control with a basal-bolus regimen significantly reduced the frequency of composite complications, including postoperative wound infection, pneumonia, bacteremia, and acute renal and respiratory failure (71). In that study, treatment with basal-bolus insulin reduced average total inpatient costs per day by 14% or $751 compared totreatment with a correction bolus dose insulin alone (133). Another study from Australia has shown that in a randomized study of 1371 surgical inpatients, 680 with even a single glucose value >200 mg/dl (11.1 mmol/l) received early intervention from the diabetes inpatient team (134). This led to reductions in glucose of a modest -5.4 mg/dl (-0.3 mmol/l), which still equated to a 4.6%, statistically significant reduction in hospital-acquired infections compared to those receiving standard care (134).

HYPOGLYCEMIA

Hypoglycemia is the most common side effect of treatment of all types of diabetes and stress hyperglycemia in thehospital setting. It presents a significant barrier to satisfactory long-term glycemic control. Hypoglycemia results from animbalance between glucose supply, glucose utilization, and current insulin levels. Hypoglycemia is defined as a lower-than-normal level of blood glucose. Hypoglycemia is defined as any glucose level <70 mg/dl (<3.9 mmol/l) (108; 135) for hospital inpatients. Level 1 hypoglycemia is a glucose concentration of 54 – 70 mg/dL (3.0 – 3.9 mmol/L). Level 2 hypoglycemia is a blood glucose concentration of <54 mg/dL (3.0 mmol/L) (108). Severe hypoglycemia has been defined by many as <40 mg/dl (<2.2 mmol/l) (136), but a newer definition, Level 3 hypoglycemia, is a glucose concentration low enough where the individual requires third-party assistance to aid recovery (108). The UK JBDS guideline suggests that the lower limit of glucose in the inpatient population should be 108 mg/dl (6.0 mmol/l), and that the range between 72 – 108 mg/dl (4.0 – 6.0 mmol/l) be designated ‘looming’ hypoglycemia, to alert ward staff to take action because of the possibility that lower glucose levels may be associated with harm (137). The exception to this is those people on a diet only or those people on an insulin pump / closed loop system who can self-manage their diabetes while in the hospital.

The incidence of severe hypoglycemia in the different trials ranged between 5% and 28%, depending on the intensity ofglycemic control in the ICU (138). Rates from subcutaneous insulin trials in non-critically ill patients range from less than 1% to 33% (71; 139; 140). In 2017, the UK National Diabetes Inpatient Audit (NaDIA) data showed that 18% of peoplewith diabetes in hospital experienced one or more hypoglycemic episodes with a blood glucose <72mg/dl (<4.0 mmol/l) –down from 26% in 2011, with 7% (1 in 14) of all inpatients experiencing episodes requiring third party assistance to administer rescue therapy (141). The NaDIA data also showed that those with type 1 diabetes had the highestprevalence, with 25% experiencing a severe hypoglycemic episode (141). Furthermore, 1.3% (1 in 80) of those in hospitals with diabetes required some form of injectable rescue treatment (i.e., IV glucose or IM glucagon), down from2.1% in 2011 (141). The same data showed that the highest proportion of episodes occurred overnight (28%) between 05:00 and 09.00 AM when snack availability was likely the lowest (140; 141).

Table 3 lists some key factors that predict the likelihood of someone experiencing a hypoglycemic event whilehospitalized. These also include older age, greater illness severity, diabetes, and the use of oral glucose-loweringmedications and/or insulin (137; 142-145). In-hospital processes of care that contribute to the risk for hypoglycemia include unexpected changes in nutritional intake that are not accompanied by associated changes in the glycemic management regimen. Examples include (but are not limited to) cessation of nutrition for procedures, an adjustment in the amount of nutritional support, interruption of the established routine for glucose monitoring, deviations from the established glucose control protocols, and failure to adjust therapy when glucose is trending down, or steroid therapy isbeing tapered (137; 146; 147). A common cause of inpatient hypoglycemia is when handwritten insulin prescriptions lead to errors, including misreading, e.g., when ‘U’ is used for units (i.e., 4U becoming 40 units) or confusing the insulin name with the dose (e.g., Humalog Mix25 becoming Humalog 25 units) (148). Electronic prescribing has been associated with a lower rate of prescription errors (141).

However, other factors may also be involved, such as concurrent use of drugs with hypoglycemic agents, e.g., warfarin, quinine, salicylates, fibrates, sulphonamides (including co-trimoxazole), monoamine oxidase inhibitors, NSAIDs, probenecid, somatostatin analogs, or selective serotonin reuptake inhibitors. Secondary causes of inpatienthypoglycemia include loss of counter-regulatory hormone function, e.g., Addison’s disease, growth hormone deficiency,hypothyroidism, or hypopituitarism.

Table 3. Common Risk Factors for Developing Hypoglycemia in the Hospital
Prior episode of hypoglycemia
Older age
Chronic kidney disease
Congestive heart failure
Liver Failure
Sepsis
Malnutrition
Erratic eating patterns / Nutritional interruptions / Lack of access to carbohydrates
Malignancies
Insulin regimen
Type 1 diabetes
Mental status changes
Certain concomitants use of medications
Duration of diabetes

The development of hypoglycemia is associated with adverse hospital outcomes (29; 30; 117; 118; 124; 144; 149-155). Turchin et al. examined data from 4,368 admission episodes for people with diabetes, of which one-third were on regular insulin therapy (30). Patients experiencing inpatient hypoglycemia experienced a 66% increased risk of death within one year and spent 2.8 days longer in the hospital compared to those not experiencing hypoglycemia. A 2019 systematic review and meta-analysis of hospital-acquired hypoglycemia in non-ICU patients suggested that adults exposed to glucose levels <72 mg/dl (<4.0 mmol/l) experienced a mean increased length of hospital stay of 4.1 days (95% CI 2.36 – 5.79) compared to those who did not experience hypoglycemia (144). The same dataset suggested an increased relative risk of in-hospital mortality for non-ICU patients of 2.09 (95% CI 1.64 – 2.67) (144). There was a non-significant reduction in mortality for those in ICU of 0.75 (95% CI 0.49 – 1.16) (144). The odds ratio (95% confidence interval) for mortality associated with one or more episodes was 2.28 (1.41-3.70, p=0.0008) among a cohort of 5,365 patients admitted to amixed medical-surgical ICU (142). In a larger cohort of over 6,000 patients, hypoglycemia was associated with longer ICU stays and greater hospital mortality, especially for patients with more than one episode of hypoglycemia (29). These data strengthen the argument to have potentially less strict glycemic targets for those not on ICU (32; 137). For example, if an individual has a glucose of 75 mg/dl (4.2 mmol/l), and is on an intravenous insulin infusion, by the time their bedside capillary glucose is next measured, they may have a glucose well below 72 mg/dl (4.0 mmol/l), thus they have come to potential harm. Indeed, data published from previous NaDIA surveys and NaDIA Harms using data from over 100 hospitals across the UK showed several serious adverse events, including seizures, permanent cerebral damage, cardiac arrests, and deaths. Insulin therapy was implicated in several of these events (33; 34; 156; 157). The counterargument is that there are initiatives to reduce the risk of developing inpatient hypoglycemia and having national guidance has led to improved patient care overall (106; 158). As with the outpatient population, the increased use of technology may help avoid hypoglycemia (159).

Hypoglycemia has been associated with adverse cardiovascular outcomes, such as increased myocardial contractility, prolonged QT interval (possibly due to the rapid drop in potassium concentrations due to the increased circulating epinephrine and norepinephrine), ischemic electrocardiogram changes and repolarization abnormalities, angina, arrhythmias, increased inflammation, and sudden death, (51; 160-162). The mechanisms for the poor outcome have yet to be entirely understood. Still, hypoglycemia has been associated with increases in pro-inflammatory cytokines (TNFα,IL-1β, IL-6, and IL-8), markers of lipid peroxidation, acute changes in endothelial dysfunction with associated vasoconstriction, increased blood coagulability, cellular adhesion, and oxidative stress (163; 164).

Despite these observations, the direct causal effect of iatrogenic hypoglycemia on outcome is still debatable. Kosiborod et al. reported that spontaneous hypoglycemia, but not insulin–induced hypoglycemia, was associated with higherhospital mortality (152). Similarly, another study among 31,970 patients also reported that hypoglycemia is associatedwith increased in-hospital mortality. Still, the risk was limited to patients with spontaneous hypoglycemia and not topatients with drug-associated hypoglycemia (165). These studies raise the possibility that hypoglycemia, like hyperglycemia, despite the biochemical and other changes described, is a marker of disease burden rather than a directcause of death.

RECOMMENDATIONS FOR MANAGING HYPERGLYCEMIA IN THE HOSPITAL ENVIRONMENT

Knowledge of Diabetes Management Amongst Medical Staff

The burden on inpatient diabetes falls most frequently on junior medical staff, who often have little or no specialist diabetes training. As such, it is perhaps unsurprising that errors occur. In the UK, a survey of junior doctors showed that unlike other commonly encountered medical conditions, such as acute asthma or angina, their knowledge about and confidence in managing diabetes was significantly lower (166). In 2019, this was also shown in a multicenter study from the US – with the major difference being that whilst most staff felt confident and comfortable managing diabetes, when challenged on how to manage certain situations, and in particular identifying glucose targets for those who were critically ill or the threshold for defining hypoglycemia, their confidence was far higher than their knowledge – a potentially devastating combination (167). Given the high prevalence of diabetes amongst hospital inpatients, essential diabetes management should be part of mandatory training. However, studies have found that despite the implementation of training programs, structured staff education has not shown to be of significant benefit in terms of improved patient outcomes (168; 169)

Management of Inpatient Hyperglycemia in the ICU

Insulin is the best way to control hyperglycemia in the inpatient setting, especially in critically ill patients. A variable-rate intravenous insulin infusion is the preferred method to achieve the recommended glycemic target (ADA Standards of Care 2025). The short half-life of intravenous insulin makes it ideal in this setting because it allows flexibility in the eventof unpredicted changes in an individual’s health, medications, and nutrition.

When someone is identified as having hyperglycemia (blood glucose ≥180 mg/dl [≥10.0 mmol/l]), a variable rate intravenous insulin infusion should be started to maintain blood glucose levels <180 mg/dl (<10.0 mmol/l). A variety ofintravenous infusion protocols are effective in achieving glycemic control with a low rate of hypoglycemic events and in improving hospital outcomes (73; 86; 113; 121; 170-174). A proper protocol should allow flexible blood glucose targets to be modified based on the individual’s clinical situation. Further, it should have clear instructions about the blood glucosethreshold for initiating an insulin infusion and the initial rate. The appropriate fluids should also be prescribed. It should bevalidated to avoid hyperglycemia if adjusted too slowly and hypoglycemia if adjusted too fast. Accurate insulin administration requires a reliable infusion pump that can deliver the insulin dose in increments of 0.1 units per hour (138; 172).

There is no ideal insulin protocol for managing hyperglycemia in the critically ill patient. In addition, no clear evidence demonstrates the benefit of one protocol/algorithm versus any other (138). Implementing any of these algorithms requires close follow-up by the nursing staff and is prone to human errors. Some institutions have developedcomputerized protocols that can be implemented to avoid errors in dosing (138; 175-179). Essential elements thatincrease protocol success of continuous insulin infusion are: 1) rate adjustment considers the current and previous glucose value and the current rate of insulin infusion, 2) rate adjustment considers the rate of change (or lack of change) from the previous reading, and 3) frequent glucose monitoring (hourly until stable glycemia is established, and then at least every 2 – 3 hours) (138; 171; 180-182).

Several computer-based algorithms aiming to direct the nursing staff in adjusting the insulin infusion rate have become commercially available (175-177; 179; 183). Retrospective cohorts and controlled trials have reported a more rapid and tighter glycemic control with computer-guided algorithms than standard paper form protocols in ICU patients (176; 184), as well as lower glycemic variability than patients treated with the standard insulin infusion regimens. Despite differences in glycemic control between insulin algorithms, another study showed no difference between computerized protocolsversus conventional glucose control (128). Thus, most insulin algorithms appear to be appropriate alternatives for managing hyperglycemia in critically ill patients, and the choice depends upon the physician’s preferences, staffing availability, and cost considerations. As mentioned, the increasing implementation of available technology, in particular the use of closed loops should improve the management of dysglycemia over the coming years (185-187).

Managing Hyperglycemia in the Non-ICU Setting

Subcutaneous insulin is the preferred therapeutic agent for glucose control in those admitted to non-ICU settings under general medicine and surgery. A recent study suggested that the use of bolus correction doses of subcutaneous insulin (“subcutaneous sliding scale insulin” (SSI)) is an acceptable way of controlling dysglycemia, particularly in those whose admission glucose levels were <180 mg/dl (10 mmol/l) (188; 189). However, many studies do not agree and advocate against using this method as the only way to control glucose levels because it results in undesirable hypoglycemia andhyperglycemia or inadequately controls dysglycemia (109; 190-193). It has become evident in recent years that the useof scheduled subcutaneous insulin therapy with basal (e.g. glargine, detemir or degludec) once daily or with intermediate-acting insulin (NPH) given twice daily alone or in combination with short (regular) or rapid-acting insulin (lispro, aspart, glulisine) prior to meals is effective and safe for the management of most patients with hyperglycemia and diabetes (20; 108; 194).

The basal-bolus (prandial) insulin regimen is considered the physiologic approach as it addresses the three componentsof insulin requirement: basal (what is required in the fasting state), nutritional (what is needed for peripheral glucosedisposal following a meal), and supplemental (what is necessary for unexpected glucose elevations, or to dispose ofglucose in hyperglycemia (195).

A prospective, randomized multi-center trial compared the efficacy and safety of a basal/bolus insulin regimen with basal-bolus regimen and SSI in people with type 2 diabetes admitted to a general medicine service (139). The use of a basal-bolus insulin regimen improved blood glucose control more than the subcutaneous sliding scale alone. A blood glucose target <140 mg/dl (<7.8 mmol/l) was achieved in 66% of those in the glargine plus glulisine group and 38% in the slidingscale group (139). The incidence of hypoglycemia, defined as a BG <60 mg/dl (<3.3 mmol/l), was less than 5% in thosetreated with basal-bolus or SSI. A different study on general surgery inpatients also compared the efficacy and safety of a basal-bolus regimen to SSI in those with type 2 diabetes (71). The basal-bolus regimen resulted in a significant improvement in glucose control and a reduction in the frequency of the composite of postoperative complications, including wound infection, pneumonia, respiratory failure, acute renal failure, and bacteremia.

Multi-dose human NPH and regular insulin have been compared to basal-bolus treatment with insulin analogs in an open-label, controlled, multicenter trial in 130 medical admissions with type 2 diabetes (196). This study found that both treatment regimens significantly improved inpatient glycemic control with a glucose target of <140 mg/dl (<7.8 mmol/l) before meals and no difference in the rate of hypoglycemic events. Thus, a similar improvement in glycemic control can be achieved with either basal-bolus therapy with insulin analogs or with NPH/regular human insulin in people with type 2diabetes.

Most people in the hospital have reduced caloric intake due to a lack of appetite, medical procedures, or surgical intervention. In the Basal Plus trial, people with type 2 diabetes who were treated with diet, oral antidiabetic agents, orlow-dose insulin (≤ 0.4 unit/kg/day) prior to admission were randomized to receive a standard basal-bolus regimen withglargine once daily and glulisine before meals or a single daily dose of glargine. In addition, supplemental doses ofglulisine were administered for correction of hyperglycemia (>140 mg/dl [>7.8 mmol/l]) per sliding scale (197). This study reported that the basal approach resulted in similar improvement in glycemic control and the frequency of hypoglycemia compared to a standard basal-bolus regimen (197). Thus, in insulin-naive individuals or those receiving low-dose insulin on admission, as well as those with reduced oral intake, the use of a basal plus regimen is an effective alternative to basal-bolus (108).

The recommended total daily insulin dose should start between 0.3 to 0.5 units per Kg (139; 147; 198; 199) for mostpeople with diabetes. Starting doses greater than 0.6 – 0.8 units/kg/day have been associated with 3-fold higher odds of hypoglycemia than doses lower than 0.2 U/kg/day. In elderly individuals or those with impaired renal function, lower initial daily doses (≤ 0.3 units/kg) may lower the risk of hypoglycemia (200).

Hospital Use of Non-Insulin Therapy in Non-Critical Care Settings

Several other classes of non-insulin glucose-lowering agents have been tried in the hospital setting. However, most are not suitable for use. Metformin, while the first line for type 2 diabetes in the outpatient setting, may not be appropriate where there is any evidence of dehydration, renal impairment, or if intravenous contrast is due to be administered due to the risk of lactic acidosis or worsening of renal function (195). Despite the lack of robust evidence of benefit, it remains in everyday use in many countries (201). Thiazolidinediones are excellent at lowering glucose but are used rarely, and possibly inappropriately, in hospitalized patients because they take several weeks to reach their maximum effect, may precipitate heart failure, and may cause peripheral edema due to fluid retention (202-204).

Sulfonylureas work rapidly and are often the drugs of choice for worsening diabetes in an outpatient setting (205). They remain in everyday use in many countries, with up to 20% of inpatients with diabetes in the USA and UK remaining on them (140; 203). However, they increase the risk of hypoglycemia. There is data to show that they remain one of the most frequent causes of inpatient hypoglycemia, thus extending the length of hospital stay and increasing the risk of inpatient mortality (141; 206-208).

Oral glucose-lowering medication use is limited by the delay and unpredictability of onset of action, and there is also concern regarding the cardiovascular effects of sulfonylureas and the contraindication of metformin use in patients with renal or liver dysfunction (19; 209). Recent work using the sodium-glucose co-transporter 2 inhibitors for corticosteroid-induced hyperglycemia in acute exacerbation of chronic obstructive pulmonary disease (COPD) or used in COVID infections failed to demonstrate an improvement in outcomes (210; 211). Indeed, despite their clear benefits in the outpatient population with and without diabetes, robust evidence for the benefit of SGLT2i use in the inpatient population (in people with diabetes) is lacking (212; 213).

The use of oral antidiabetic agents was not recommended in previous guidelines because of the need for more safety and efficacy studies in the inpatient setting (20). However, increasing evidence indicates that treatment with dipeptidyl peptidase-4 (DPP4) inhibitors, alone or in combination with basal insulin, is safe and effective in general medicine and surgery with mild to moderate hyperglycemia (48). In a pilot study, general medicine and surgical inpatients with bloodglucose between 140 and 400 mg/dl (7.8 – 22.2 mmol/l) treated with diet, oral antidiabetic drugs, or low-dose insulin (≤0.4 U/kg/day) were randomized to sitagliptin once daily, sitagliptin and basal insulin, or basal-bolus insulin (214). All groups received correction doses of lispro before meals and bedtime for blood glucose >140 mg/dl (>7.8 mmol/l). In those with mild-moderate hyperglycemia (<180 mg/dl [<10 mmol/l]), the use of sitagliptin plus supplemental (correctiondoses) or in combination with basal insulin resulted in no significant differences in mean daily blood glucose, frequency of hypoglycemia or the number of treatment failures compared to the basal-bolus regimen (214). The SITA-HOSPITAL trial, a multicenter, randomized controlled study in 279 general medicine and surgery individuals with type 2 diabetes previously treated with oral anti-diabetic agents or low-dose insulin (<0.6 U/kg/d), also reported similar glycemic control,hypoglycemia rate, hospital length-of-stay, treatment failures or hospital complications (including acute kidney injury orpancreatitis) between the combination of oral sitagliptin plus basal insulin to the more labor-intensive basal-bolus insulin regimen (215).

Analysis from prospective studies using DPP4-i in various inpatient situations with type 2 diabetes (T2D) reported that treatment with DPP4-i alone or with basal insulin suggested they were safe and lowered glucose concentrations without increasing the risk of hypoglycemia (216; 217).

For people with type 2 diabetes hospitalized with heart failure, the ADA has recommended that the use of a sodium-glucose cotransporter 2 (SGLT2) inhibitor be initiated or continued during hospitalization and upon discharge if there are no contraindications and after recovery from the acute illness (218-221). In patients with acute heart failure, empagliflozin was well tolerated, resulting in significant clinical benefits, including heart failure readmissions and quality of life (221). However, SGLT2 inhibitors should be avoided in cases of severe illness, in people with type 1 diabetes, ketonemia, or ketonuria, and during prolonged fasting and surgical procedures. Proactive adjustment of diuretic dosing is recommended during hospitalization and/or discharge, especially in collaboration with a cardiology/heart failure consult team. The FDA has warned that SGLT2 inhibitors should be stopped three days before scheduled surgeries (4 days in the case of ertugliflozin) (222). This differs from the UK guideline, which states that these drugs should be omitted from the day before a procedure (223).

Staffing Levels in the Hospital

Inadequate levels of appropriately knowledgeable staff are a concern for patients with diabetes (224). An insufficient level of specialist diabetes staff is a factor that inhibits safe and optimal care (34). Recently, the UK JBDS group developed a simple calculator into which individual teams could enter data to calculate their staffing needs (49). That data showed the discrepancy between the number of people delivering care and the number of people that specialist teams felt was necessary to provide safe and effective care for five days or seven days per week. This was for senior medical staff, specialist nursing staff, dieticians, podiatrists, pharmacists and psychologists (49).

Glucose Monitoring in the Hospital

All patients admitted to the hospital with a diagnosis of diabetes and those with newly discovered hyperglycemia should be monitored closely (21). The frequency and method of monitoring and the schedule of the blood glucose checks will depend on the nutritional intake, patient treatment, and insulin schedule, as well as the ability of the individual to self-manage their diabetes (225). There is some controversy regarding the best method to monitor blood glucose. However,considering the convenience and wide availability of capillary point of care (POC) testing, we suggest this as the bestapproach if done with a monitoring device that has demonstrated accuracy (226-228). When using POC blood glucose meters, it is important to keep several things in mind. In particular, overall clinical conditions that might affect the POC value, such as hemoglobin level, perfusion, and medications, as well as the policy of the health care organization in guiding the patient and the staff on the use of POC devices or newer technologies.

Bedside point-of-care (POC) capillary glucose testing is usually ordered before meals and bedtime to assess glycemic control and adjust insulin therapy in the hospital (19; 228). However, this approach has been shown to fail to detect hypoglycemia, particularly nocturnal and asymptomatic hypoglycemia, which is a common scenario in the hospital setting (229; 230).

Continuous glucose monitoring (CGM) has increased over the last few years, helping to improve glycemic management in the ICU. The use of this technology was accelerated during the COVID pandemic, where the use of CGM meant that close contact with sick individuals was avoided using remote sensing (231-234). The use of CGM is questioned, with the accuracy of readings when dealing with hypoglycemia or in the operating room (235; 236). However, in general, most studies have been associated with overall benefit (234; 237; 238).

CGM is reliable compared to point-of-care testing and laboratory values in the inpatient setting. It is currently being evaluated for managing ICU and general ward patients (159; 185; 235; 239-242). Studies have shown that CGM offers advantages over intermittent capillary monitoring in the ICU. CGM can help identify and prevent severe hyperglycemia and hypoglycemia by allowing for more rapid and accurate adjustments to insulin infusions compared to capillary blood glucose testing. Research has also demonstrated that CGM is better at detecting hypoglycemia, predominantly asymptomatic and nocturnal hypoglycemia, than capillary glucose testing (243; 244). Additionally, CGM is as safe and effective as standard care in hospitalized patients and can lead to a significant decrease in recurrent hypoglycemia events compared with standard point-of-care testing (243; 245). Regulatory approval for CGM use in hospitals is still pending, but consensus guidelines suggest that the use of CGM in the hospital setting has the potential to provide a better glycemic assessment than capillary glucose testing (Walia et al.; other, Endo Soc Guidelines). Furthermore, advanced technology in guiding insulin therapy using machine learning and artificial intelligence is being integrated more frequently into diabetes care (246). A proof-of-concept trial in patients with type 2 diabetes evaluated the efficacy and safety of a model-based reinforcement learning framework in titrating insulin dosing. After applying the intervention, the mean daily BG was lower by approximately 56 mg/dl (3 mmol/l) with no severe hypo- or hyperglycemia (247).

The American Diabetes Association (ADA) and UK JBDS recently recommended that people with diabetes who use a personal continuous glucose monitoring (CGM) device should be allowed to continue during hospitalization (48; 159; 248). Both organizations also recommend that confirmatory point-of-care (POC) glucose measurements be used for insulin dosing decisions, hypoglycemia assessment, and treatment.

A recent survey of inpatient teams across the UK showed significant variations in accessing and using technologies (249). These included networked glucose and ketone meters, and wearable diabetes technologies such as CGM, pumps, or closed loop systems. While almost two-thirds of respondents agreed that technology would help prevent hypoglycemia, there was a wide variety of specialist diabetes nursing or medical staff support available to help non-specialists, particularly on weekends or outside of regular working hours (249).

Medical Nutrition Therapy (MNT) in Hospitalized Patients with Diabetes

Medical nutrition therapy (MNT) is a key component of the comprehensive management of diabetes and hyperglycemia in the inpatient setting. Maintaining adequate nutrition is essential for glycemic control and to meet adequate caloric demands. Caloric demand in acute illness will differ from that in the outpatient setting. Achieving the proper nutritionalbalance in the inpatient setting is challenging. Anyone admitted to the hospital with diabetes or hyperglycemia should beassessed to determine the need for a modified diet to meet caloric demands.

The general approach to addressing MNT in the inpatient setting is usually based on expert opinions and patients' needs. Limited data exist regarding the best approach or method to achieve the ideal caloric supply. To determine the best approach, method, and caloric needs of their patients, providers should work closely with the nutrition professional.

All patients with diabetes or hyperglycemia should receive an individualized assessment. Most patients will generallyreceive adequate caloric needs with 3 discrete meals daily. Further, the metabolic need for patients with diabetes isusually provided by 25 to 35 calories/kg, whereas some critically ill patients might require less than 15 to 25 calories/kg per day (250; 251). A consistent carbohydrate meal-planning system might help to facilitate glycemic control and insulin dosing in the inpatient setting. Most patients require 1,500-2000 calories daily with 12-15 grams of carbohydrates per meal (19). Ideally, the carbohydrates should come from low glycemic index foods such as whole grains and vegetables.

Those individuals unable to achieve these goals should be evaluated to determine the need for enteral or parenteral nutrition. Enteral nutrition is the second-best option after oral nutrition and should be preferred over parenteral nutrition in hospitalized individuals (252-254). There are several advantages of enteral feeding versus parenteral feeding, including low cost, low risk of complications, a physiologic route, less risk for gastric mucosa atrophy, and lower risk of infectiousand thrombotic complications compared with the latter form of therapy (252-254). The benefit of parental nutrition has been documented in critically ill patients. However, some research has shown a detrimental effect on patients withdiabetes and hyperglycemia. Parental nutrition should be considered only in patients who cannot receive enteral nutritionand should be coordinated with the institution’s parenteral nutrition team. There has been guidance published in the surgical population on peri-operative nutrition, but the recommendations for people with diabetes is lacking because the literature remains scanty (251). A recent UK survey of diabetes teams showed no consensus on enteral feeding regimens (253). For those tube-fed, there were 3 main regimens: continuous 24-hour feeding, a single feed with one break in 24 hours, or multiple feeds in 24 hours. In addition, there were multiple insulin regimens used: premixed insulin, isophane insulin, analog basal insulin, variable rate intravenous insulin, or basal-bolus insulin. None of these provided adequate glycemic control (253).

Enteral and parenteral nutrition can prevent the effects of starvation and malnutrition (252). Enteral nutrition over parenteral nutrition is preferred whenever possible due to a lower risk of infectious and thrombotic complications (254-256). Standard enteral formulas reflect the reference values for macro- and micronutrients for a healthy population and contain 1-2 cal/ml. Most standard formulas contain whole protein, lipids in the form of long-chain triglycerides, andcarbohydrates. Standard diabetes-specific formulas provide low amounts of lipids (30% of total calories) combined with a high carbohydrate (257) content (55–60% of total calories); however, newer “diabetic” formulas have replaced part of carbohydrates with monounsaturated fatty acids (up to 35% of total calories) and also include 10-15 g/l dietary fiber andup to 30% fructose (257; 258).

“Diabetic” enteral formulas containing low-carbohydrate high–monounsaturated fatty acid (LCHM) are preferable to standard high-carbohydrate formulas in hospitalized patients with type 1 and type 2 diabetes (257; 258). In a meta-analysis of studies comparing relatively newer enteral LCHM formulas with older formulations, the postprandial rise inblood glucose was reduced by 18- 29 mg/dl [1.0-1.6 mmol/l] with the newer formulations (258). Table 4 depicts the composition of standard and diabetic-specific enteral formulas commonly used in hospitalized patients.

Table 4. Composition of Standard and Diabetic Specific Enteral Formulas Commonly Usedin Hospitalized Patients in the USA
	Calories(kcal/mL)	Carbohydrate(g/l)		Fat (g/l)	Protein (g/l)	Manufacture
Standard formula
Jevity^® 1.0 Cal	1.0	140		35	40	Abbott Nutrition
Nutren^® 1.0	1.0	109		27	70	Nestle Nutrition
Osmolite^® 1.2 Cal	1.2	158		39	56	Abbott Nutrition
Jevity^® 1.2	1.2	169		39	56	Nestle Nutrition
Fibersource^® HN	1.2	164		40	54	Nestle Nutrition
Isosource^® 1.5 Cal	1.5	176		60	68	Nestle Nutrition
Jevity^® 1.5	1.5	216		50	64	Nestle Nutrition
Diabetes specific formula
Glucerna^® 1.0 Cal	1.0	75	54		50	Abbott Nutrition
Nutren^® Glytrol®	1.0	100	48		45	Nestle Nutrition
Glucerna^® 1.2 Cal	1.2	114	60		60	Abbott Nutrition
Diabetisource^® AC	1.2	100	59		60	Nestle Nutrition
Glucerna® 1.5 Cal	1.5	133	75		83	Abbott Nutrition

The UK Joint British Diabetes Societies has updated its guidelines for the management of diabetes in enterally fed people (259).

Corticosteroid Therapy – Impact on Blood Glucose

Steroid use in hospitalized patients is common. A single-center cross-sectional study showed that 12.8% of all the people in the hospital were on glucocorticoids (260). Steroids may be administered by various regimes and at variable doses. A single daily dose of steroid (e.g., prednisolone/prednisone) in the morning may be the most standard mode ofadministration (205; 260-262). Limbachia et al. showed that, in susceptible individuals, steroid use will often result in arise in blood glucose by late morning that continues through to the evening (263). Overnight, the blood glucose generallyfalls back to baseline levels by the following day. They also showed the differential effects between different steroid types, with oral or IV dexamethasone or methylprednisolone leading to higher glucose excursions than prednisolone or hydrocortisone (262). Thus, treatment should be tailored to treating the hyperglycemia while avoiding nocturnal and earlymorning hypoglycemia. Multiple daily doses of steroid, be it intravenous hydrocortisone or oral dexamethasone, can cause a hyperglycemic effect throughout the 24-hour period. It may be, however, that a twice-daily premixed or basal-bolus regimen may need to be started if oral medication or once-daily insulin proves insufficient to control hyperglycemia (205). Close attention will therefore need to be paid to blood glucose monitoring, and early intervention may benecessary.

Glucose levels in most individuals can be predicted to rise approximately 4 to 8 hours following the administration of once-daily oral steroids and sooner following the administration of intravenous steroids. Again, capillary blood glucose monitoring is paramount to guide appropriate therapeutic interventions. Conversely, glucose levels may improve to pre-steroid levels 24 hours after intravenous steroids are discontinued. When oral steroids are weaned down, the glucose levels may decline in a dose-dependent fashion, but this may not occur, particularly in those with pre-existing undiagnosed diabetes.

At the commencement of steroid therapy, or for those already on a supraphysiological dose of corticosteroid, capillary blood glucose testing should occur before meals and at bedtime, in particular before lunch or evening meal, when thehyperglycemic effects of a morning dose of steroid are likely to be greatest (205; 262).

Subcutaneous insulin using a basal or multiple daily injection regimen will likely be the most appropriate choice for most patients to achieve glycemic control in the event of hyperglycemia. While the UK has advocated for short-acting sulfonylureas (205), the morning administration of basal human insulin may closely fit the glucose excursion induced by a single morning dose of oral steroid. Basal analog insulin may be appropriate if hyperglycemia is present for more prolonged periods. However, if long-acting insulin analogs are used in this context, care should be taken to identify and protect against hypoglycemia overnight and in the early morning. Subsequent titration of the insulin dose may be required to maintain glucose control in the face of increasing or decreasing the steroid dose.

When a patient is discharged from the hospital on steroid therapy, a clear strategy for managing hyperglycemia or potential hyperglycemia and the titration of treatment to address the hyperglycemia should be communicated to the community diabetes team and primary care team. Patients who commenced on steroids as inpatients and were discharged after a short stay with the intention of continuing high-dose steroids should receive standard diabetes education, encompassing the risks associated with hyperglycemia and hypoglycemia.

Closed Loop Technology

Several organizations have recommended that people who are well enough to do so should continue to use their insulin pumps in hospitals (108; 109; 240; 264). However, only a few recent studies have reported using closed-loop systems, also referred to as the artificial pancreas or automated insulin delivery systems, in hospitalized inpatients. Small randomized trials have reported good efficacy with improved time in target and lower mean daily blood glucose without an increased rate of hypoglycemia in the ICU (265-267) and non-ICU settings (268-271). However, some of these studies were done in those with type 2 diabetes (268; 270). In one non-ICU study, the time in the target range between 100-180 mg/dl (5.6-10.0 mmol/l) was reported as 59.8% in patients using the closed-loop technology compared to 38.1% with standard subcutaneous insulin regimen (269).

Similarly, a closed-loop study in patients receiving nutritional support also reported higher time in target glucose (68% vs 36.4%) and lower mean glucose values (153 vs 205 mg/dl [8.5-11.4 mmol/l]) compared to a standard insulin regimen (270). As with the use of CGM in the hospital, treatment with artificial pancreas is still experimental, and larger studies are needed to prove its safety and efficacy in ICU and non-ICU settings. Further challenges lie ahead because of the unfamiliarity of these systems, with non-specialist staff the primary carers for people with diabetes.

The ADA has recommended that insulin pumps or automated insulin delivery (closed-loop) systems be continued for hospitalized individuals with diabetes when clinically appropriate. Confirmatory POC blood glucose measurements should be used for insulin-dosing decisions and for assessing and treating hypoglycemia. However, this depends on the availability of required supplies and resources, proper training, ongoing competency assessments, and the implementation of institutional diabetes technology protocols (48).

As with the CGM, those who are well and can self-manage can look after their devices and diabetes. However, in those who are unwell or incapacitated, the systems must be disengaged from automatic and set to ‘manual’ mode to allow the diabetes teams to help manage the diabetes. The systems may not also be able to cope with the acute changes that occur in the hospital, including (but not limited to) change in oral carbohydrate intake, the use of glucocorticoids or other medications inducing insulin resistance; peri-operative use, nausea and vomiting; enteral or parenteral nutrition. Once again, the use of ‘manual’ mode is recommended in these situations, and the diabetes is managed in conjunction with the specialist diabetes team.

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Assessing Insulin Sensitivity and Resistance in Humans

ABSTRACT

In this chapter we discuss a representative variety of methods currently available for estimating insulin sensitivity/resistance. These range from complex, time consuming, labor-intensive, invasive procedures to simple testsinvolving a single fasting blood sample. It is important to understand the physiological concepts informing each method so that relative merits and limitations of particular approaches are appropriately matched with proposed applications and data is interpreted correctly. The glucose clamp method is the reference standard for direct measurement of insulin sensitivity. Regarding simple surrogates, QUICKI and Log (HOMA) are among the best and most extensively validated. Dynamic tests are useful if information about both insulin secretion and insulin action are needed.

INTRODUCTION

Insulin resistance plays a major pathophysiological role in type 2 diabetes and is tightly associated with major public health problems including obesity, hypertension, coronary artery disease, dyslipidemias, and a cluster of metabolic and cardiovascular abnormalities that define the metabolic syndrome (1, 2, 3).

A global epidemic of obesity is driving the increased incidence and prevalence of type 2 diabetes and its cardiovascular complications (4). Insulin resistance is commonly associated with visceral adiposity, glucose intolerance, hypertension, dyslipidemia, hypercoagulable state, endothelial dysfunction, and/or elevated markers of inflammation. Therefore, the presence of these clinical abnormalities is usually characteristic of an insulin resistant state. In addition to clinical manifestations of the “Insulin Resistance Syndrome,” insulin resistance predisposes to accelerated cardiovascular disease (CVD). Therefore, it is of great importance to develop tools for quantifying insulin sensitivity/resistance in humans that may be used to appropriately investigate the epidemiology, pathophysiological mechanisms, outcomes of therapeutic interventions, and clinical course of patients with insulin resistance (5). In this chapter, we will discuss some currently used methods for assessing insulin sensitivity, their applications, merits, and limitations.

INSULIN SENSITIVITY AND RESISTANCE

Metabolic actions of insulin help to maintain glucose homeostasis and promote glucose utilization (6). Insulin increases glucose utilization in peripheral organs (e.g., skeletal muscle and adipose tissue) and suppresses hepatic glucose production (HGP) and adipose tissue lipolysis. In addition to these classical metabolic target tissues, insulin has many other important physiological targets. These include the brain, pancreatic β-cells, heart, and vascular endothelium that help to coordinate and couple metabolic and cardiovascular homeostasis under healthy conditions (6-9). Insulin has concentration- dependent saturable actions to increase whole-body glucose disposal. The maximal effect of insulin defines “insulin responsiveness” while the insulin concentration required for a half-maximal response defines “insulin sensitivity” (Fig. 1). Although, other actions of insulin on fat and amino-acid metabolism, cardiovascular, kidney, and brain function also exhibit a concentration-dependent response, the term “insulin sensitivity” typically refers to insulin’s metabolic actions to promote glucose disposal.

The concept of insulin resistance was proposed as early as 1936 to describe diabetic patients requiring high doses of insulin (10). Insulin resistance is typically defined as decreased sensitivity and/or responsiveness to insulin- mediatedglucose disposal and/or inhibition of HGP and adipose tissue lipolysis. Rigorous evaluation of altered sensitivity and responsiveness therefore requires a comparison of insulin dose-response curves.

Figure 1. Schematic representation of concentration-response relationships between plasma insulin concentrations and insulin-mediated whole-body glucose disposal. Curve a: normal insulin sensitivity and responsiveness. Curve b: rightward shift in insulin concentration-response curve. This represents decreased insulin sensitivity (increased EC50) with normal insulin responsiveness. Curve c: Decreased insulin sensitivity (increased EC50) and reduced insulin responsiveness. Curve d: Leftward shift in the insulin concentration- response curve. This represents increased insulin sensitivity (decreased EC50) with normal insulin responsiveness.

DIRECT MEASURES OF INSULIN SENSITIVITY

Hyperinsulinemic Euglycemic Glucose Clamp

PROCEDURE

The glucose clamp technique, originally developed by Andres and DeFronzo is widely accepted as the reference standard for directly determining metabolic insulin sensitivity in humans (11). After an overnight fast, insulin is infused intravenously at a constant rate that may range from 5 - 120 mU/m²/min (dose per body surface area per minute). This constant insulin infusion results in a new steady-state insulin level that is above the fasting level (hyperinsulinemic). As a consequence, glucose disposal in skeletal muscle and adipose tissue is increased while HGP is suppressed. Under these conditions, a bedside glucose analyzer is used to frequently monitor blood glucose levels at 5 – 10 min intervals while 20% dextrose is given intravenously at a variable rate in order to “clamp” blood glucose concentrations in the normal range (euglycemic). An infusion of potassium phosphate is also given to prevent hypokalemia resulting from hyperinsulinemia and increased glucose disposal. After several hours of constant insulin infusion, steady-state conditions are typically achieved for plasma insulin, blood glucose, and the glucose infusion rate (GIR). Assuming that the hyperinsulinemic state is sufficient to completely suppress hepatic glucose production, and since there is no net change in blood glucose concentrations under steady- state clamp conditions, the GIR must be equal to the glucose disposal rate (M) (Fig. 2). Thus, whole body glucose disposal at a given level of hyperinsulinemia can be directly determined. M is typically normalized to body weight or fat-free mass to generate an estimate of insulin sensitivity.Alternatively, an insulin sensitivity index derived from clamp data can be defined as SIClamp = M/(G x ΔI), where M is normalized for G (steady-state blood glucose concentration) and ΔI (difference between fasting and steady-state plasma insulin concentrations) (12).

Figure 2 Schematic representation of the “steady state” dynamics of glucose and insulin during an euglycemic hyperinsulinemic glucose clamp.

The validity of glucose clamp measurements of insulin sensitivity depends on achieving steady-state conditions. “Steady-state” is often defined as a period greater than 30- min (at least 1 h after initiation of insulin infusion) during which the coefficient of variation for blood glucose, plasma insulin, and GIR are less than 5% (12, 13). It is possible to use stable isotope or radio-labeled glucose tracer under clamp conditions to estimate HGP so that appropriate corrections can be made to M in the event HGP is not completely suppressed (14, 15, 16,17). An alternative approach is to use an insulin infusion rate sufficiently high to completely suppress HGP according to the insulin sensitivity/resistance of the population to be studied. M is routinely obtained at only a single insulin infusion rate and therefore comparisons between M or SIClamp among different subjects is valid only if the same insulin infusion rate is used for all subjects. When glucose tracers are used during a clamp study, the tracer is infused at constant rate throughout the study. HGP estimated during the last 20 or 30 min of the clamp is a measure of insulin- mediated suppression of HGP, an estimate of hepatic insulin sensitivity. Similarly, lipolytic rates can be assessed at baseline and hyperinsulinemia during clamp by using isotopic tracers (e.g., palmitate). A single or multistep hyperinsulinemic euglycemic clamp can be used to measure adipose tissue insulin sensitivity. The linear relationship between log transformed rates of palmitate flux and plasma insulin concentrations provides an IC50 (pmol/L) for suppression of lipolysis (18).

ADVANTAGES AND LIMITATIONS

The principal advantage of the glucose clamp in humans is that it directly measures whole body glucose disposal at a given level of insulinemia under steady-state conditions. Conceptually, the approach is straightforward and there are a limited number of assumptions which are clearly defined. In research settings where assessing insulin sensitivity/resistance is of primary interest and feasibility is not an issue (e.g., study population < 100) it is appropriate to use the reference standard glucose clamp technique. The main limitations of the clamp approach are that it is time-consuming, labor intensive, expensive, and requires an experienced operator to manage technical difficulties. Thus, for epidemiological studies, large clinical investigations, or routine clinical applications (e.g., following changes in insulinresistance after therapeutic intervention in individual patients) application of the glucose clamp is not feasible. Nevertheless, when measured in relatively large cohorts, the M values showed a bimodal pattern, with an optimal cutoff of 5 mg/min/kg-FFM using a 40 mU/min·m². However, IR was defined as a glucose disposal rate below 4.9 mg/min/kg, using a 120 mU/min·m² dose (80, 81).

Insulin-Suppression Test (IST)

PROCEDURE

The insulin-suppression test, another method that directly measures metabolic insulin sensitivity/resistance, was introduced by Shen et. al. in 1970 and subsequently modified by Harano et. al. (19, 20). After an overnight fast, somatostatin (250 μg/h) or the somatostatin analogue octreotide (25 µg bolus, followed by 0.5 µg/min) (21) is intravenously infused to suppress endogenous secretion of insulin and glucagon. Simultaneously, insulin (25 mU/m²/min) and glucose (240 mg/m²/min) are infused into the same antecubital vein over 3 h. From the contralateral arm, blood samples for glucose and insulin determinations are taken every 30 min for 2.5 h and then at 10 min intervals from 150 - 180 min of the IST. The constant infusions of insulin and glucose determine steady-state plasma insulin (SSPI) andglucose (SSPG) concentrations. The steady-state period is assumed to be from 150 - 180 min after initiation of the IST. SSPI concentrations are generally (but not always) similar among subjects. Therefore, the SSPG concentration will be higher in insulin resistant subjects and lower in insulin sensitive subjects. That is, SSPG values are inversely related to insulin sensitivity. The IST provides a direct measure (SSPG) of the ability of exogenous insulin to mediate disposal of an intravenous glucose load under steady-state conditions where endogenous insulin secretion is suppressed.

ADVANTAGES AND LIMITATIONS

The SSPG is a highly reproducible direct measure of metabolic actions of insulin that is less labor-intensive and less technically demanding than the glucose clamp. Indeed, since there are no variable infusions with the IST, steady-state conditions are more easily achieved with the IST than with the glucose clamp. Estimates of insulin sensitivity determined by SSPG correlate well with reference standard glucose clamp estimates in normal subjects (r = 0.93) and in patients with type 2 diabetes mellitus (r = 0.91). (22, 23). Indeed, SSPG has positive predictive power for cardiovascular disease events and onset of type 2 diabetes (24, 25). In research settings where assessing insulin sensitivity/resistance is of primary interest and feasibility is not an issue, it is appropriate to use the IST. Moreover, the IST can be used for larger populations that may pose difficulties for application of the glucose clamp (26). Many of the limitations of the IST are similar to those described above for the glucose clamp (with the exception that the IST is less technically demanding).Thus, it is impractical to apply the IST in large epidemiological studies or in the clinical care setting. SSPG under ideal conditions determines primarily skeletal muscle insulin sensitivity and is not designed to reflect hepatic insulin sensitivity.

INDIRECT MEASURES OF INSULIN SENSITIVITY

Minimal Model Analysis of Frequently Sampled Intravenous Glucose Tolerance Test (FSIVGTT)

PROCEDURE

The minimal model, developed by Bergman, Cobelli, and colleagues in 1979, provides an indirect measure of metabolic insulin sensitivity/resistance based on glucose and insulin data obtained during an FSIVGTT (27). After an overnight fast, an intravenous bolus of glucose (0.3 g/kg body weight) is infused over 2 min starting at time 0. Currently, a modified FSIVGTT is used where exogenous insulin (4 mU/kg/min) is also infused over 5 min beginning 20 min after the intravenous glucose bolus (28, 29,30). Some studies use tolbutamide instead of insulin in the modified FSIVGTT to stimulate endogenous insulin secretion (15, 29, 31, 32, 27). Blood samples are taken for plasma glucose and insulinmeasurements at -10, -1, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 22, 23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100,120, 160, and 180 min. These data are then subjected to minimal model analysis using the computer program MINMOD to generate an index of insulin sensitivity (SI).

The minimal model is defined by two coupled differential equations with four model parameters (Fig. 3). The first equation describes plasma glucose dynamics in a single compartment. The second equation describes insulin dynamics in a “remote compartment”. The structure of the minimal model allows MINMOD to uniquely identify model parameters that determine a best fit to glucose disappearance during the modified FSIVGTT. SI is calculated from two of these model parameters and is defined as fractional glucose disappearance per insulin concentration unit. In addition to SI, other minimal model parameters may be used to estimate a “glucose effectiveness” index (SG). SG is defined as the ability of glucose per se to promote its own disposal and inhibit HGP in the absence of an incremental insulin effect (i.e., when insulin is at basal or constant concentrations).

Recently the minimal model has been used to assess free fatty acid (FFA) insulin sensitivity. Using a one compartment nonlinear model of FFA kinetics during FSIVGTT, showed that the FFA insulin sensitivity parameter correlated well with minimal model indices (33). Furthermore, this model also showed that glucose modulates disposal of FFAs.

Figure 3. Schematic equations, and parameters for the minimal model of glucose metabolism. Differential equations describing glucose dynamics (G(t)) in a monocompartmental “glucose space” and insulin dynamics in a “remote compartment” (X(t)) are shown at the top. Glucose leaves or enters its space at a rate proportional to the difference between plasma glucose level, G(t) and the basal fasting level, Gb. In addition, glucose also disappears from its compartment at a rate proportional to insulin levels in the “remote” compartment (X(t)). In this model, t = time; G(t) = plasma glucose at time t; I(t) = plasma insulin concentration at time t; X(t) = insulin concentration in “remote” compartment at time t; Gb = basal plasma concentration; Ib = basal plasma insulin concentration; G(0) = G0 (assuming instantaneous mixing of the IV glucose load); p1, p2, p3, and G0 = unknown parameters in the model that are uniquely identifiable from FSIVGTT; glucose effectiveness, SG = p1; and insulin sensitivity, SI = p3/p2.

ADVANTAGES AND LIMITATIONS

Minimal model analysis of the modified FSIVGTT is easier than the glucose clamp method because it is slightly less labor intensive, steady-state conditions are not required, and there are no intravenous infusions that require constant adjustment. Unlike the glucose clamp or IST, information about insulin sensitivity, glucose effectiveness, and β-cellfunction can be derived from a single dynamic test. The minimal model generates excellent predictions of glucose disappearance during the FSIVGTT. SI is a strong predictor of the development of diabetes in a prospective study of children of diabetic parents (34). Moreover, the insulin-modified FSIVGT may be used in relatively large- scale population studies (35). Therefore, in research settings where assessing insulin sensitivity along with glucose effectiveness and β-cell function is of interest, minimal model analysis of the insulin-modified FSIVGTT may be appropriate. The minimal model approach is simpler than direct methods for determining insulin sensitivity. Nevertheless, it still involves intravenous infusions with multiple blood sampling over a 3 h period that is nearly as labor intensive as the glucose clamp or IST. In addition, many limitations of minimal model analysis stem from the fact that the model oversimplifies the physiology of glucose homeostasis and is discussed in detail elsewhere (5).

Oral Glucose Tolerance Test (OGTT)

The oral glucose tolerance test (OGTT) is a simple test widely used in clinical practice to diagnose glucose intolerance and type 2 diabetes (36). After overnight fast, blood samples for determinations of glucose and insulin concentrations are taken at 0, 30, 60, and 120 min following a standard 75g oral glucose load. Oral glucose tolerance reflects the efficiency of the body to dispose of glucose after an oral glucose load or meal. The OGTT mimics the glucose and insulin dynamics of physiological conditions more closely than conditions of the glucose clamp, IST, or FSIVGTT. However, it is important to recognize that glucose tolerance and insulin sensitivity are not equivalent concepts. In addition to metabolic actions of insulin, insulin secretion, incretin effects, and other factors contribute importantly to glucose tolerance. Thus, the OGTT and meal tolerance tests provide useful information about glucose tolerance but not insulin sensitivity/resistance per se.

Intravenous and Oral Tracer Studies

The use of tracers for estimation of insulin sensitivity was first introduced in 1986 to overcome the shortcomings of FSIVGTT (37) The minimal model method does not allow segregation of glucose production from liver from exogenously administered glucose during calculations of insulin sensitivity and thus induces error in the insulin sensitivity calculations. Labeled intravenous glucose can be differentiated from endogenously produced glucose and thus use of labeled glucose during IVGTT provides more precise and accurate measurements (38,39) Similarly, labeled glucose has been used in oral glucose tolerance test and insulin sensitivity has been calculated by minimal model technique similar toFSIVGTT(40). There is a strong correlation of insulin sensitivity calculated from labeled oral minimal model with insulin sensitivity calculated from gold standard euglycemic hyperinsulinemic clamp, r=0.7, p<0.001 (41). There are dual tracer and triple tracer methods as well to estimate the hepatic/endogenous glucose production and discussion of these methods is beyond the scope of this review (42). Basal hepatic insulin resistance index can then be estimated as the product of HGP rate and the fasting plasma insulin concentration. Use of tracer definitely allows for improvement over the FSIVGTT. Use of labeled oral glucose allows for more precise measurements of insulin sensitivity and glucose disposal from a simple OGTT and this can be a useful tool in large studies. The triple tracer method is cumbersome and cannot be employed in large studies.

SIMPLE SURROGATE INDEXES FOR INSULIN SENSITIVITY/RESISTANCE

Surrogates Derived from Fasting Steady-state Conditions

PROCEDURE

After an overnight fast, a single blood sample is taken for determination of blood glucose and plasma insulin. In healthy humans, the fasting condition represents a basal steady-state where glucose is homeostatically maintained in the normal range such that insulin levels are not significantly changing and HGP is constant. That is, basal insulin secretion by pancreatic β cells determines a relatively constant level of insulinemia that will be lower or higher in accordance with insulin sensitivity/resistance such that HGP matches whole body glucose disposal under fasting conditions. Surrogate indexes based on fasting glucose and insulin concentrations reflect primarily hepatic insulin sensitivity/resistance. However, under most conditions, hepatic and skeletal muscle insulin sensitivity/resistance are proportional to each other. In the diabetic state with fasting hyperglycemia, fasting insulin levels are inappropriately low and insufficient to maintain euglycemia. Therefore, definitions of the more useful surrogate indexes take these considerations into account. Due to lack of a standardized insulin assay, it is not possible to use surrogate indexes to define universal cutoff points for insulin resistance.

ADVANTAGES AND LIMITATIONS

Simple surrogate indexes of insulin sensitivity/resistance are inexpensive quantitative tools that can be easily applied in almost every setting including epidemiological studies, large clinical trials, clinical research investigations, and clinical practice. If a direct measure of insulin sensitivity is not required, not feasible to obtain, or if insulin sensitivity is ofsecondary interest, it may be appropriate to use a surrogate index. The relative merits and limitations of individual surrogate indexes are discussed below.

The Homeostasis Model Assessment (HOMA)

HOMA, developed in 1985, is a model of interactions between glucose and insulin dynamics that is then used to predict fasting steady-state glucose and insulin concentrations for a wide range of possible combinations of insulin resistance and β-cell function (43). The model assumes a feedback loop between the liver and β-cell (43, 44, 45); glucose concentrations are regulated by insulin- dependent HGP while insulin levels depend on the pancreatic β-cell response to glucose concentrations. Thus, deficient β-cell function reflects a diminished response to glucose-stimulated insulinsecretion. Likewise, insulin resistance is reflected by diminished suppressive effect of insulin on HGP. HOMA model describes this glucose-insulin homeostasis by a set of empirically derived non-linear equations. The model predicts fasting steady- state levels of plasma glucose and insulin for any given combination of pancreatic β-cell function and insulin sensitivity. Computer simulations have been used to generate a grid from which mathematical transformations of fasting glucose and insulin β-cell function (HOMA %B) from steady-state conditions. An important caveat for HOMA is that it imputes dynamic β-cell function (i.e., glucose-stimulated insulin secretion) from fasting steady- state data. In the absence of dynamic data, it is difficult, if not impossible, to determine the true dynamic function of β-cell insulin secretion.

In practical terms, most studies using HOMA employ an approximation described by a simple equation to determine a surrogate index of insulin resistance. This is defined by the product of the fasting glucose and fasting insulin divided by a constant. Thus, HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mmol/l) / 22.5. The constant is a normalizing factor, the product of fasting plasma insulin of 5 µU/mL and plasma glucose of 4.5 mmol/L obtained from an “ideal” and “normal” individual. Therefore, for an individual with normal insulin sensitivity, HOMA-IR = 1. It is important to note that over wide ranges of insulin sensitivity/resistance Log (HOMA-IR), (which normalizes the skewed distribution of fasting insulinvalues) determines a much stronger linear correlation with glucose clamp estimates of insulin sensitivity (12). HOMA orLog (HOMA) is extensively used in large epidemiological studies, prospective clinical trials, and clinical research studies (45, 46, 47). In research settings where assessing insulin sensitivity/resistance is of secondary interest or feasibility issues preclude the use of direct measures by glucose clamp, it may be appropriate to use Log (HOMA-IR). However, as discussed below, other surrogate indexes have certain advantages over HOMA or Log (HOMA) in some circumstances.

Quantitative Insulin Sensitivity Check Index (QUICKI)

QUICKI is an empirically-derived mathematical transformation of fasting blood glucose and plasma insulin concentrations that provides a reliable, reproducible, and accurate index of insulin sensitivity with excellent positive predictive power (12, 48,13, 49, 50). Since fasting insulin levels have a non-normal skewed distribution, log transformation improves its linear correlation with SIclamp. However, as with 1/(fasting insulin) and the G/I ratio, this correlation is not maintained in diabetic subjects with fasting hyperglycemia and impaired β-cell function that is insufficient to maintain euglycemia. To accommodate these clinically important circumstances where fasting glucose is inappropriately high and insulin is inappropriately low, addition of log (fasting glucose) to log (fasting insulin) provides a reasonable correction such that the linear correlation with SIClamp is maintained in both diabetic and non-diabetic subjects. The reciprocal of this sum results in further transformation of the data generating an insulin sensitivity index that has a positive correlation with SIclamp. Thus, QUICKI = 1/Log (Fasting Insulin, µU/ml) + Log (Fasting Glucose, mg/dl). Over a wide range of insulin sensitivity/resistance, QUICKI has a substantially better linear correlation with SIclamp (r ≈ 0.8 – 0.9) than SI derived from the minimal model or HOMA-IR (12, 48, 49). Log (HOMA) is roughly comparable to QUICKI in this regard. Multiple independent studies find excellent linear correlations between QUICKI and glucose clampestimates (either GIR or SIClamp) in healthy subjects, obesity, diabetes, hypertension, and many other insulin- resistant states (49, 51, 52, 53, 54, 55, 56). QUICKI is among the most thoroughly evaluated and validated surrogate index for insulin sensitivity. As a simple, useful, inexpensive, and minimally invasive surrogate for glucose clamp-derived measures of insulin sensitivity, QUICKI is appropriate and effective for use in large epidemiological or clinical researchstudies, to follow changes after therapeutic interventions, and for use in studies where evaluation of insulin sensitivity is not of primary interest.

Adipose Tissue Insulin Resistance Index (Adipo-IR)

Adipo-IR is a measure similar to HOMA-IR in that it is obtained from a fasting level of FFA and insulin (product of FFA and insulin levels). Recent studies have shown that Adipo-IR correlates well with the gold standard measure of adipose tissue insulin sensitivity derived from one-step hyperinsulinemic-euglycemic clamp technique using a palmitate tracer (57). Age and physical fitness were however shown to affect the predictive values. Thus, Adipo-IR may be suitable for larger population studies, however the multistep pancreatic clamp technique is probably needed for mechanistic studies of adipose tissue insulin action.

Surrogates Derived from Dynamic Tests

PROCEDURE

Surrogate indexes of insulin sensitivity that use information derived from dynamic tests include OGTT, meal tolerance tests, and IVGTT. Procedures for these tests have been described in a previous section. Specific indexes including Matsuda index (58), Stumvoll index (59), Avignon index (60), oral glucose insulin sensitivity index (OGSI) (61), Gutt index (62), and Belfiore index (63) use particular sampling protocols during the OGTT or the meal. In addition, minimal model approaches have been used to model plasma glucose and insulin dynamics during an OGTT or a meal to determine insulin sensitivity/resistance (64). Glucose disposal of an oral glucose load or a meal is mediated by a complex dynamic process that includes gut absorption, glucose effectiveness, neurohormonal actions, incretin actions, insulin secretion, and metabolic actions of insulin that primarily determine the balance between peripheral glucose utilization and HGP. Surrogate indexes that depend on dynamic testing take into account both fasting steady-state and dynamic post-glucose load plasma glucose and insulin levels. After an oral glucose challenge, the HGP is maximally suppressed for approximately 60 min and remains suppressed at a constant level for the subsequent 60–120 min time period. Therefore, glucose uptake by peripheral tissues (e.g., muscle and adipose tissue) primarily determines the rate ofdecrease in plasma glucose concentration from its peak value to its nadir during an OGTT. Based on this observation, surrogate indices of hepatic and muscle insulin sensitivity/ resistance from an OGTT has been widely used (65). Recent studies comparing the OGTT- derived, tissue-specific surrogate indices hepatic insulin resistance index (HIRI) andmuscle insulin sensitivity index (MISI) with clamp measurements showed that surrogate indices derived from an OGTT are accurate in predicting insulin sensitivity but are not tissue-specific (66). Studies using oral tracers in OGTT, with measurement of insulin sensitivity from OGTT and then comparing these to clamp measurements, would be crucial to ascertain the validity these measures. Indeed, recent studies have shown that it is possible to measure hepatic insulin sensitivity in healthy volunteers and in prediabetes with the use of single tracer (67).

ADVANTAGES AND LIMITATIONS

Many surrogate measures derived from dynamic data correlate reasonably well with glucose clamp estimates of insulin sensitivity (58, 61,62). Estimates of insulin sensitivity derived from OGTT predict the development of type 2 diabetes in epidemiologic studies ( 50, 68, 65). The advantage of surrogates based on dynamic testing is that information about insulin secretion can be obtained at the same time as information about insulin action. However, if one is only interested in estimating insulin sensitivity/resistance, fasting surrogates may be preferable to dynamic surrogates because they are simpler to obtain. The oral route of glucose delivery is more physiological than intravenous glucose infusion. However, poor reproducibility of the OGTT and meal tolerance test due to variable glucose absorption, splanchnic glucose uptake, and additional incretin effects need to be considered. Thus, distinguishing direct metabolic actions of insulin following oral ingestion of glucose or a mixed meal is more problematic than after FSIVGTT. In addition, as with many other measures of insulin sensitivity, surrogates derived from dynamic testing generally incorporate both peripheral and hepatic insulin sensitivity. Although OGTT involves considerably less work than FSIVGTT, dynamic testing in general requires more effort and cost than fasting blood sampling.

ETHNIC DIFFERENCES

Hispanics, African Americans, and South Asians are highly prone to develop diabetes. A meta-analysis showed that non-diabetic Africans have lower insulin sensitivity and higher insulin response after an intravenous glucose load comparedto Caucasians and East Asians ( 69). In a study that compared euglycemic hyperinsulinemic clamp derived glucosedisposal rates (GDR) with HOMA-IR, QUICKI, and OGTT-derived indices, fasting insulin levels and HOMA-IR did not correlate with GDR whereas Matsuda index derived from OGTT significantly correlated with GDR in African American men (70). Similarly, in another study in Afro-Caribbean adults, HOMA-IR did not correlate with insulin sensitivity calculated from FSIVGTT and M-value calculated from hyperinsulinemic euglycemic clamp (71). Likewise, IR predictive ability of QUICKI and HOMA-IR was limited in Asian-Indian men (72). Recent studies highlight that minimal model may underestimate insulin sensitivity between groups when acute insulin response (AIR) is higher in one group (73). African Americans have reduced insulin clearance and higher AIR than Whites, suggesting that the minimal model may underestimate insulin sensitivity in African Americans (73). These studies suggest that at least in some ethnic groups, QUICKI and HOMA-IR may only be useful as secondary outcome measurements in assessing insulin sensitivity/resistance and studies inferring lower insulin sensitivity in non- diabetic African Americans based on FSIVGTT and minimal modeling should be interpreted cautiously.

METABOLOMICS

Metabolomics is an interrogation and quantification of small-molecule metabolites in body fluids and tissues. It aims at identifying and quantifying small molecules in the sample by either using mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy. The details of the methodology and its application in diabetes research are beyond the scope of this chapter. In this chapter, we will focus on new markers of insulin resistance that have been discovered using this approach. Using a non- targeted approach, Gall et al. metabolically profiled fasting plasma samples from 399 non-diabetic, clinically healthy subjects (74). Insulin sensitivity was measured using euglycemic hyperinsulinemic clamps. Individuals in the bottom tertile of the cohort were designated as insulin- resistant. Among the 485 candidate biomarkers identified, plasma α-hydroxybutyrate levels were inversely related to insulin sensitivity and this association was independent of age, sex and BMI. Other metabolites such as linoleoyl- glycerophosphocholine (L-GPC), glycine, and creatine were also highly correlated with insulin sensitivity. Using 26 metabolites from this study, the group went on to develop a model called Quantose algorithm to predict insulin resistance. Fasting insulin, α-hydroxybutyrate, L- GPC and oleate levels were included in this model. Quantose IR as a fasting surrogate of insulin sensitivity was superior to other simple surrogate measures and was able to predict the progression from normal glucose tolerance to impaired glucose tolerance (75). Branched chain amino acids (BCAAs) were found to significantly increase in obese compared to lean subjects and a BCAA based index correlated with HOMA (76). The elevation of BCAA in subjects with impaired fasting glucose and diabetes has been confirmed in subsequent studies (77).

Lipoprotein insulin resistance score (LPIR) is a novel metabolomic biomarker based on nuclear magnetic resonance (NMR) quantification of lipoprotein levels and sizes. This index has been shown to predict future type 2 diabetes mellitus is some cohorts (78). LPIR is derived from the weighted score of six lipoproteins (VLDL, LDL, and HDL sizes and concentrations) that are more strongly related to IR than each of its individual subclasses (79). A risk score of between0-100 is estimated, with a score of 100 denoting being most insulin resistant. These metabolomic studies are promising since they can measure hundreds of metabolites in a very small sample. However, the pricing, technology, and access, precludes its use clinically. Further studies using this approach are necessary in larger more heterogeneous cohorts to replicate and validate surrogate insulin resistance markers derived through metabolomics

Table 1. Methods for Assessing Insulin Sensitivity and Resistance in Humans
Method	Measure of Insulin sensitivity
Direct Measures
Hyperinsulinemic Euglycemic Glucose Clamp	Average glucose infusion rate (GIR) = glucose disposal rate (M). SIClamp = M/(G x ΔI), where M is normalized for G (steady-state blood glucose concentration) and ΔI (difference between fastingand steady-state plasma insulin concentrations)
Insulin-suppression Test (IST)	Steady-state plasma glucose (SSPG) concentrations duringconstant infusions of insulin and glucose with suppressed endogenous insulin secretion
Indirect Measures
Minimal Model Analysis of Frequently Sampled IntravenousGlucose Tolerance Test (FSIVGTT)	Minimal model uniquely identifies model parameters thatdetermine a best fit to glucose disappearance during the modified FSIVGTT. SI : fractional glucose disappearance per insulin concentration unit; SG (glucose effectiveness): ability of glucose per se to promote its own disposal and inhibit HGP in the absenceof an incremental insulin effect (i.e., when insulin is at basal levels).
Simple Surrogate Indexes
Surrogates Derived from Fasting Steady-state Conditions
The Homeostasis Model Assessment (HOMA)	HOMA-IR = [(Fasting Insulin (µU/mL)) X (Fasting Glucose(mmol/L))]/22.5
Quantitative Insulin Sensitivity Check Index (QUICKI)	QUICKI = 1/[Log (Fasting Insulin, µU/ml) + Log (Fasting Glucose,mg/dl)]
Surrogates Derived from Dynamic Tests (OGTT)
Matsuda Index	ISI(Matsuda) = 10000/√[(Gfasting (mg/dl) x Ifasting (µU/ml) x(Gmean x Imean)]
Gutt Index - ISI (0, 120) (mg.l2.mmol-1.mIU-1.min-1)	ISI (0, 120) = 75000 + (G0-G120)(mg/l) x 0.19 x BW / 120 xGmean (0, 120min) (mmol/l) x Log [Imean (0, 120min)](mU/l)

Gmean, mean plasma glucose concentration during OGTT; Go, plasma glucose concentration during fasting; G120, plasma glucose concentration at 120 min; Gmean, mean plasma glucose concentration during OGTT; Imean, mean insulin concentration during OGTT; Io, plasma insulin concentration during fasting; I120, plasma insulin concentration at 120 min.

SUMMARY

In this chapter we have discussed a representative variety of methods currently available for estimating insulin sensitivity/resistance (but this is by no means an exhaustive review) (Table 1). These range from complex, timeconsuming, labor-intensive, invasive procedures to simple tests involving a single fasting blood sample. It is important to understand the physiological concepts informing each method so that relative merits and limitations of particular approaches are appropriately matched with proposed applications and data is interpreted correctly. The glucose clamp method is the reference standard for direct measurement of insulin sensitivity. Regarding simple surrogates, QUICKI and Log (HOMA) are among the best and most extensively validated. Dynamic tests are useful if information about both insulin secretion and insulin action are needed.

ACKNOWLEDGEMENTS

This work was supported by the Intramural Research Program, NIDDK, NIH

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Physiology of GnRH and Gonadotropin Secretion

ABSTRACT

Gonadotropin hormone-releasing hormone (GnRH) is the key regulator of the reproductive axis. Its pulsatile secretion determines the pattern of secretion of the gonadotropins, follicle stimulating hormone and luteinizing hormone, which then regulate both the endocrine function and gamete maturation in the gonads. Recent years have seen rapid developments in how GnRH secretion is regulated, with the discovery of the kisspeptin-neurokinin-dynorphin neuronal network in the hypothalamus. This mediates both positive and negative sex steroid feedback control of GnRH secretion, in conjunction with other neuropeptides and neurotransmitters. This chapter describes the main features of this regulatory system, including how its anatomical arrangements interact with functional control, and describes key differences between rodent and larger mammalian systems.

INTRODUCTION

Since the discovery of Gonadotropin Releasing Hormone (GnRH), an extensive body of literature has established it as the pivotal central regulator of human reproduction. However, the GnRH neuronal network, per se lacks the cellular machinery to fully integrate developmental, environmental, endocrine, and metabolic factors that influence its secretion. For example, GnRH neurons do not express the principal estrogen receptor alpha (ER-alpha), which is required for sex-steroid mediated control of gonadotropin secretion (1). Intermediate signaling pathways must therefore exist to mediate gonadal steroid feedback. Current evidence, accumulated since the discovery of Kisspeptin-Neurokinin B-Dynorphin (KNDy) neuronal network in the last decade, suggests a pivotal role for this network in the regulation of pulsatile GnRH secretion by integrating nutrient, endocrine, and environmental signals, and thus the control of downstream hypothalamic-pituitary-gonadal (HPG) axis.

The HPG axis anatomically comprises:

The hypothalamus (especially the infundibular nucleus, the human homologue of the arcuate nucleus) where the KNDy and GnRH-producing neurons are located.
The anterior pituitary, where Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH) are secreted by gonadotropes.
The gonads, responsible for the production of both sex steroids and gametes, under the influences of LH and FSH.

As with other endocrine systems, positive and negative feedback regulate HPG axis (2,3). In this chapter, we have focused on human data. Where human data is limited, data from other species are leveraged.

GONADOTROPIN RELEASING HORMONE (GnRH) – THE PRINCIPAL REGULATOR OF REPRODUCTION

The Discovery of GnRH

GnRH was isolated from porcine hypothalami and structurally identified as a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly·NH2) five decades ago (4-6). This decapeptide was shown to potently stimulate LH and FSH release from the pituitary in a number of mammalian species (6,7). Early literature referred to this peptide as the ‘Luteinizing Hormone-Releasing Hormone (LH-RH)’, but more recently, it is widely referenced as Gonadotropin Releasing Hormone (GnRH) -reflecting the stimulatory role on the secretion of both gonadotropins – i.e., LH and FSH (8).

Diverse forms of GnRH and its receptor exist among vertebrates, with over twenty primary structures across species, suggesting that GnRH system developed early in the evolutionary sequence (9,10). The GnRH structure was first identified in mammals and is therefore referred to as GnRH I (9,11). Subsequently, another structurally different vertebrate GnRH sequence was first identified from chicken brain -this is now referred to as GnRH II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2) (10,12). A third form has also been described in fish - GnRH III (9,12). In mammals, hypophysiotropic functions are limited to GnRH I, therefore in the human context GnRH I is referred to as GnRH (13) and we will use this terminology for this review.

Neuroanatomy of GnRH Neurons

GnRH neurons originate in the medial olfactory placode during embryological development and migrate along the olfactory bulb to their final positions within the hypothalamus (14,15). A number of factors contributing to this GnRH neuron migratory process have been identified: anosmin-1 (the product of KAL gene) (16), neuropilins (17), leukemia inhibitory factor (18), fibroblast growth factor receptor 1 (19), fibroblast growth factor receptor 8 (20), polysialic form of neural adhesion molecule (PSA-NCAM) (21), among others (22). Defective GnRH migration leads to Kallmann syndrome, characterized by hypogonadotropic hypogonadism due to GnRH deficiency and anosmia (15). Mutations in prokineticin genes (PROK1 and PROK2) lead to hypogonadotropic hypogonadism without anosmia, suggesting that factors other than suboptimal migration can also lead to functional deficiencies in GnRH (15,23,24).

GnRH cell bodies are located in the medial preoptic area (POA) and in the arcuate/infundibular nucleus of the hypothalamus, forming a neuronal network with projections to the median eminence (25). GnRH is secreted from the median eminence into the fenestrated capillaries of portal circulation, carried to the anterior pituitary (25). In humans, the number of GnRH neurons has been estimated to range between 1000 to 1500 (9,14). The co-location of GnRH neurons with other central regulators allows the GnRH network to be influenced by a range of neuroendocrine and metabolic inputs (26).

GnRH Secretion and Pulsatility

Two distinct modes of GnRH secretion have been described: pulsatile and surge modes (26). Pulsatile mode refers to episodic release of GnRH, with distinct pulses of GnRH secretion into the portal circulation with undetectable GnRH concentrations between pulses. The surge mode of GnRH secretion occurs in females, during the pre-ovulatory phase, in which the presence of GnRH in the portal circulation appears to be persistent (26,27).

Direct pulsatile GnRH release was initially demonstrated in ovariectomized rhesus monkeys using serial samples of portal blood (28). Pulsatile pattern of GnRH secretion was demonstrated subsequently in humans through serial blood sampling during pituitary surgery (29). Abolishment of LH pulses by GnRH antisera (30,31) and its reestablishment with GnRH analogues (30) suggest that LH pulses are determined by the underlying GnRH pulsatility. The LH pulsatility was first detected during an attempt to validate a radioimmunoassay to measure serum LH in rhesus monkeys, where marked variations in LH levels was noticed (32). Further studies confirmed the pulsatile nature of LH secretion (33-35). In women, the frequency and amplitude of LH pulses were noted to be dependent on the menstrual cycle phase, with pulses every 1 to 2 hours during the early follicular phase eventually merging into a continuous mid-cycle surge, and decreased pulse frequency to every 4 hours during the luteal phase (36). In humans, LH pulse frequency is used as a surrogate of GnRH pulsatility, as ethical considerations and technical challenges preclude sampling of hypophyseal blood or cerebrospinal fluid to measure GnRH concentrations directly (37,38).

The importance of GnRH pulsatility on LH and FSH secretion was first demonstrated in rhesus monkeys, where endogenous GnRH secretion was abolished by hypothalamic radiofrequency. Pulsatile GnRH reinstated gonadotropin secretion in these animals, whereas continuous GnRH only elicited a transient response. Moreover, the switch from continuous to pulsatile GnRH administration allowed recovery of gonadotropin secretion (39).

GnRH neurons coordinate their activity, but the precise mechanism of this remains unclear (27,40), and is the subject of continuing investigations. Episodic multi-unit electrical activity at medial basal hypothalami (MBH) is correlated with LH release, suggesting that ‘GnRH pulse generator’ is anatomically located at MBH – or closely linked to it neurohormonally(41,42). GnRH neurons show intrinsic electrical pulsatility. GnRH cell lines derived from mouse hypothalamic and fetal olfactory placode GnRH neurons both demonstrate intrinsic pulsatility in vitro (26,43,44). Functionally, the ‘GnRH pulse generator’ relies on complex relations between glutamatergic cells, GnRH and other neurons, and likely other elements are involved, of which the kisspeptin-neurokinin B-opioid pathway may have a pivotal intermediary role in the regulation of GnRH pulsatility (45).

Differential Regulation of LH and FSH

The stimulatory effects of GnRH on LH and FSH secretion are not identical (46). FSH secretion is more irregular than LH in both humans and sheep, which is essentially related to the pulsatility and different stimulatory effects of GnRH, but other factors also might be relevant, such as differences in LH and FSH storage (more scarce for the FSH), existence of different gonadotropes subpopulations, or diverse response times to GnRH (47). In ovariectomized sheep administered GnRH antisera, pulsatile secretion of LH was completely inhibited (undetectable LH levels within 24 hours), while the FSH concentration fell more slowly and remained detectable (30). It has been estimated that 93% of the GnRH pulses were associated with FSH pulses and, unlike LH, a constitutive secretion of FSH appears to exist (48). The frequency of GnRH input has been demonstrated to selectively regulate gonadotropin subunit gene transcription: rapid GnRH pulse rates increase α and LH-β and slow GnRH pulse frequency increases FSH-β gene transcription (49-51). Moreover, with progressive increases in GnRH frequency (from one pulse every 120 to 60 min, from 60 to 30 min, and from 30 to 15 min) in GnRH deficient men, mean LH rose concurrently with a decrease in LH pulse amplitude, while FSH remained unchanged (52).

Biological and Clinical Relevance of GnRH Pulsatility

Appropriate modulation of LH pulse frequency is essential for pubertal maturation and reproductive function. In infancy, LH pulsatile secretion is increased (often termed mini-puberty), likely reflecting pulsatile GnRH secretion, but soon becomes quiescent (53). This pre-pubertal suppression of HPG axis has been shown to occur in agonadal humans (54)and primates (55), suggesting that hypothalamo-hypophyseal factors play a role in post-natal quiescence of the reproductive axis, until puberty sets in.

The onset of pubertal maturation is heralded by the development of a pattern of steady acceleration in LH pulsatility (56). In children, higher basal and GnRH stimulated LH concentrations are observed in early childhood (<5 years). This is subdued mid-childhood (5-11 years) and increase thereafter with pubertal development (54,57). Conceptually, an abnormal reactivation of GnRH pulse frequency is the central mechanism associated with precocious or delayed puberty (14).

In women, the pattern of GnRH secretion is essential for the regulation of the menstrual cycle (Figure 1) (58,59). LH pulse frequency is slow in the luteal phase, and increasingly speeds up during the follicular and the pre-ovulatory phases, presumably reflecting changes in GnRH pulse frequency (60). Abnormalities in GnRH - and hence LH pulse frequency - are associated with a number of reproductive endocrine disorders. In hypothalamic amenorrhea, a condition associated with anovulatory amenorrhea and hypoestrogenism, LH pulse frequency (and by inference GnRH) is lower than expected for the prevailing steroid profile and is comparable to luteal phase pulsatility (37). LH pulse frequency in hyperprolactinemic women is also lower than in healthy women, requiring dopaminergic agonist preparations, such as bromocriptine to regulate prolactin secretion and restore LH pulse frequency (38). In polycystic ovary syndrome LH pulse frequency and amplitude are higher throughout the menstrual cycle in comparison to that observed in healthy women, contributing to chronic anovulation (61-64).

Figure 1. Hormonal oscillations through the menstrual cycle. In the early follicular phase of the menstrual cycle, the initial increase in FSH stimulates follicular recruitment and maturation. The consequent secretion of estradiol (E2) selectively inhibits FSH release (needed for selection of the dominant follicle) and maintains rapid GnRH pulsatility during the late follicular phase. The persistent rapid GnRH pulses increase LH, which further stimulates E2 secretion, culminating in positive E2 feedback to produce the mid-cycle LH surge. During the LH surge, GnRH levels appear to be consistently elevated and remain elevated as LH declines, suggesting that the frequency of GnRH pulse has become very rapid or continuous, which results in desensitization of LH secretion (possibly the mechanism to terminate the LH surge). After ovulation, luteinization of the ruptured follicle results in progesterone secretion which reduces the frequency of GnRH pulses. With the demise of corpus luteum, E2, progesterone and inhibin levels fall, and the GnRH pulse frequency increases, leading to follicular maturation in the next cycle. (Adapted from: Marshall JC, Dalkin AC, Haisenleder DJ, Paul SJ, Ortolano GA, Kelch RP. Gonadotropin-releasing hormone pulses: regulators of gonadotropin synthesis and ovulatory cycles. Recent Prog Horm Res. 1991;47:155-187).

NEURONAL REGULATION OF GnRH SECRETION: THE KISSPEPTIN-NEUROKININ B-DYNORPHIN (KNDy) NEURONAL NETWORK

Whilst the central role attributed to GnRH remains undisputed, its effective function requires input from other neuronal networks. For instance, the absence of estrogen receptor alpha (ER-alpha) expression on GnRH neurons suggests the need for an intermediate signaling pathway to mediate gonadal steroid feedback (1). The discovery of kisspeptin signaling in neuroendocrine regulation of human reproduction revolutionized the current understanding of the HPG axis. Kisspeptin signaling pathway is increasingly recognized as essential for normal puberty, gonadotropin secretion, and regulation of reproduction (65-67). Other relevant kisspeptin roles have been identified such as regulation of sexual and social behavior, emotional brain processing, mood, audition, olfaction, metabolism, body composition, cardiac function, among others (68-74).

Discovery of KNDy Neuronal Network

KiSS1, the gene encoding kisspeptins, was first described in 1996 as a suppressor of metastasis in human malignant melanoma (75,76). This gene was discovered in Hershey and named in accordance with the famous chocolates ‘Hershey’s Kisses’; the inclusion of ‘SS’ is indicative of ‘suppressor sequence’. The KiSS1 gene maps to chromosome 1q32 and includes four exons of which the first two are not translated (77). The gene encodes the precursor 145 amino acid peptide, which is cleaved down to a 54 amino acid peptide. This peptide can be truncated to 14, 13 and 10 amino-acid peptides, all sharing the C-terminal sequence (78,79). These peptides are collectively referred to as kisspeptins - and Kp-10, Kp-13, Kp-14 and Kp-54 are suggested abbreviations for human kisspeptins (80). In 2001, kisspeptins were identified as ligands for the orphan G–protein receptor 54 (GPR54) (81-83), currently named KISS1R (80). KISS1R is localized to human chromosome 19p13.3 and it has five exons, encoding a 398-amino acid protein with seven trans-membrane domains (79,82). Upon binding by kisspeptin, KISS1R activates phospholipase C and recruits intracellular messengers, inositol triphosphate and diacylglycerol, which in turn lead to the release of calcium and activation of protein kinase C (82-84).

A reproductive role for kisspeptin in humans became apparent from patients with pubertal disorders which were associated with KISS1R mutations (85-87). A number of inactivating mutations of Kiss1 and Kiss1r have since been reported in animal models with phenotypes characterized by pubertal delay (88). An activating mutation in KISS1R has been described in a girl with precocious puberty: when compared to cells with wild-type transfected GPR54, cells with this mutation showed prolonged inositol phosphate accumulation and phosphorylation of extracellular signal–regulated kinase, suggesting extended activation of intracellular signaling by the mutant GPR54 (89). Missense mutations have also been reported in KISS1 gene in three unrelated children with central precocious puberty (90). Functional studies of these mutant peptides demonstrated higher resistance to in vitro degradation but normal affinity to KISS1R, thus suggestive of increased bioavailability as the mechanism by which these abnormal kisspeptins induce precocious puberty (90).

Recently in an Asian cohort, potentially regulatory polymorphisms, as rs5780218 and rs12998, in KiSS1 gene were significantly associated to genetic susceptibility to central precocious puberty in Chinese girls by single-locus analysis (91). Nevertheless, these findings are inconsistently reported in literature and require additional validation in functional studies.

A role for neurokinin B in the hypothalamic regulation was also demonstrated when genetic studies in patients from consanguineous families with hypogonadotropic hypogonadism were found to have missense mutations in TAC3 (encodes neurokinin B) and TACR3 (encodes neurokinin B receptor) (92). Other cases have been reported since (93-96).

There is also long-standing evidence for the role of opioid systems in reproduction. In 1980, Wilkes reported the localization of β endorphin in the human hypothalamus (97). Studies involving the administration of naloxone and naltrexone (opioid antagonists) to humans showed stimulatory effects on LH secretion (98,99), and other studies supported the notion that endogenous opioids play a role in the control of HPG axis (100-104). In 2007, it was demonstrated that dynorphin and kisspeptin are co-localized along with neurokinin B in the same hypothalamic neuronal population in sheep, therefore termed KNDy (Kisspeptin-Neurokinin B-Dynorphin) neurons, highlighting the possible interconnection between these neuropeptides in the control of GnRH and gonadotropin secretion (105-107). The co-localization of kisspeptin, neurokinin B and dynorphin has also been demonstrated in humans (108).

Kisspeptin neurons have also other important neuroanatomical relationships, such as with neuronal nitric oxide synthase neurons as demonstrated in prepubertal female sheep (109), or with somatostatin neurons in the rat hypothalamus (110).

Neuroanatomy of KNDy Neuronal Network

The location of kisspeptin neurons is different between rodents and human species. In humans, kisspeptin neurons are distributed in the rostral Pre-optic Area (POA) and in the infundibular nucleus in the hypothalamus (Figure 2) (108,111). In both male and female autopsy samples, the majority of kisspeptin cell bodies are identified in the infundibular nucleus, and a second dense population of kisspeptin neurons in the rostral POA (108). The infundibular nucleus (arcuate nucleus in non-human species) is similar across species, but the rostral region is more species specific (108,112,113). In rodents, the rostral population is located in the anteroventral periventricular nucleus (AVPV) and the periventricular nucleus (PeN), the continuum of this region named as the rostral periventricular region of the third ventricle (RP3V) (112,114). Humans and ruminants lack this well-defined RP3V population of kisspeptin neurons, which are more scattered within the preoptic region (113,115).

Kisspeptin axons form dense plexuses in the human infundibular stalk, where the secretion of GnRH occurs (108). Axo-somatic, axo-dendritic, and axo-axonal contacts between kisspeptin and GnRH axons were demonstrated at this level, showing that kisspeptin and GnRH networks are in close proximity (108,116). Moreover, GnRH neurons express Kiss1rmRNA, reinforcing the notion of kisspeptin involvement in GnRH secretion (117-119).

Figure 2. Neuroanatomy of kisspeptin-GnRH pathway and the control of HPG axis in humans and rodents. Kisspeptin signals directly to GnRH neurons, which express KISS1R. The location of kisspeptin neurons within the hypothalamus is species specific, residing within the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus in rodents, and within the preoptic area (POA) and the infundibular nucleus in humans. Kisspeptin neurons in the infundibular nucleus (humans)/arcuate nucleus (rodents) co-express neurokinin B and dynorphin (KNDy neurons), which autosynaptically regulate kisspeptin secretion (via neurokinin B receptor and kappa opioid peptide receptor). In humans, infundibular KNDy neurons relay negative (red) and positive (green) feedback, whereas in rodents the negative and positive steroid feedback are mediated via arcuate nucleus and AVPV respectively. The role of human POA kisspeptin neurons in sex steroid feedback is not yet clear. (Adapted from: Skorupskaite K, George JT, Anderson RA. The kisspeptin-GnRH pathway in human reproductive health and disease. Human Reproduction Update. 2014;20:485-500).

Three-quarters of kisspeptin-immunoreactive cells in the human infundibular nucleus of the hypothalamus co-express neurokinin B and dynorphin (KNDy neurons) (108,120). KNDy neurons in rodents and ruminants are localized in the arcuate nucleus of the hypothalamus. However, neurokinin B and dynorphin are absent from kisspeptin neurons in the hypothalamic POA (Figure 2) (67,115). This differential expression of neuropeptides may reflect distinct functions of these two kisspeptin populations with kisspeptin neurons in the AVPV acting as LH surge generators, while those in the ARC (including KNDy neurons) acting as LH pulse generators.

Significant kisspeptin expression was also demonstrated in central extra-hypothalamic sites, including in limbic and paralimbic brain regions, such as medial amygdala, cingulate, globus pallidus, hippocampus, putamen and thalamus, key areas of neurobiological control of sexual and emotional behaviors (reviewed in detail in (121)), as well as peripherally in organs like ovary, testis, uterus and placenta where the kisspeptin system may also play a part in reproduction function (122,123).

Apart from reproductive and central kisspeptin expression, the kisspeptin signaling system has been demonstrated in several peripheral tissues, namely, in pancreas (involved in glucose-stimulated insulin secretion); in endothelial cells of different vascular beds as coronary artery, aorta and umbilical vein (triggering vasoconstriction); in the kidney, namely, in tubular cells, collecting duct cells and vascular smooth muscle cells (involved in function and renal morphogenesis); as well as in bone, fat and liver tissue (78,124,125).

Interactions Between Kisspeptin, Neurokinin B and Dynorphin

KDNy neurons act synergistically to induce coordinated and pulsatile GnRH secretion by regulating the neuroactivity of other KDNy cells. This is supported by the existence of neurokinin B and kappa opioid peptide receptors (receptor for dynorphin) within the KNDy cells, but not kisspeptin receptors, which are predominantly expressed on GnRH neurons (107,120,126). Neuron-neuron and neuron-glia communications via gap junctions contribute for the synchronized activities among KNDy neurons (127).

Neurokinins (A and B) are members of the tachykinin family of peptides, which stimulate three related GPCRs (encoded by TACR1, TACR2 and TACR3) (128) This family also contains substance P, neuropeptide K, neuropeptide γ, hemokinin-1, and more recently endokinins. Neurokinin B acts predominantly on TACR3. Neurokinin B increases the membrane potential of KNDy neurons, leading to an increase in KNDy neuron pulsatile activity which, in turn, will promote the secretion of kisspeptin leading, ultimately, to GnRH secretion (67,129,130). Neurokinin B signaling regulates GnRH/LH secretion in healthy women, and it is crucial for the mediation of the estrogenic positive and negative feedback on LH secretion (131-133). There is rapidly increasing interest in the therapeutic value of neurokinin antagonists in several indications in reproductive health, recently reviewed in (134).

In women with polycystic ovary syndrome, the relationship between kisspeptin and gonadotropin levels has been widely explored in those with anovulatory cycles (135-138). Most studies have shown higher serum levels of kisspeptin and LH when the oligomenorrhea phenotype is present, despite the high heterogeneity observed. In this context, potential treatments targeting neuroendocrine dysfunction emerged. The administration of neurokinin 3 receptor antagonists markedly reduced serum LH concentration and pulse frequency, as well as serum testosterone (139-141). A recent study confirmed a complex crosstalk between neurokinin B and kisspeptin pathways in the regulation of GnRH secretion in polycystic ovary syndrome. In this study, kisspeptin-10 infusion given to women with polycystic ovary syndrome increased LH secretion with a direct relationship to estradiol exposure. Neurokinin 3 receptor antagonism reduced LH secretion and pulsatility, and whilst LH response to kisspeptin-10 was preserved, its relationship with circulating estradiol was not. More interestingly, although kisspeptin-10 increased LH pulse frequency, changes in other parameters of LH secretory pattern were prevented when co-administered with neurokinin 3 receptor antagonists (141).

In postmenopausal women, seven day treatment with neurokinin 3 receptor antagonist decreased LH secretion, but not FSH secretion, as well as lead to a remarkable reduction in hot flushes (142). Neurokinin 3 receptor antagonism efficiency in treating menopausal hot flushes has been also demonstrated in other clinical trials (143,144). Fezolinetant administrated in a single daily dose regimen (30 or 45mg/daily) for treatment of moderate to severe vasomotor symptoms reduced these symptoms by over 50% from baseline within the first week and persistently during the 52-week treatment period, and is now approved for the treatment of menopausal vasomotor symptoms (145-148).

A second comparable drug, elinzanetant, which differs in its pharmacology in that it is an antagonist at the NK1 as well as NK3 receptor, has also been recently demonstrated to reduce the severity and frequency of moderate-to-severe vasomotor symptoms and also to improve sleep quality and menopause-related quality of life (149). The importance of NK1 receptor antagonism in these effects is unclear.

In healthy men, neurokinin B signaling display a central role for the reproductive function, and this is functionally upstream of kisspeptin-mediated GnRH secretion: LH, FSH and testosterone secretion decreased during the administration of a neurokinin 3 receptor antagonist, while kisspeptin-10 administration restored LH secretion to the same degree before and during neurokinin 3 receptor antagonist treatment (150).

An increase in the expression of Kiss1 in the hypothalamic neurons was observed following senktide (agonist of neurokinin B) administration (151), and its stimulatory effects were abolished in Gpr54 knock-out male (152). In ovariectomized goats, neurokinin B stimulated LH secretion through electrical multi-unit activity corresponded to LH secretion, suggesting a hypothalamic site for this GnRH pulse generation (153). GnRH antagonists abolished the stimulatory effect of neurokinin B, demonstrating its site of action to be functionally higher than the GnRH receptor (154,155).

Dynorphins act as the decelerator that inhibits KNDy neurons pulsatility. Studies involving the administration of opioid antagonists to humans have shown stimulatory effects on LH secretion in late follicular and mid-luteal phase (98,99), and together with other studies (100-104), highlight the inhibitory input by dynorphins on kisspeptin signalling, and consequently on GnRH/gonadotropin secretion. Through the stimulatory effects of neurokinin B and kisspeptin, and the inhibitory action of dynorphin, these neuropeptides coordinate pulsatile GnRH and LH secretion (Figure 2) (156,157).

Kisspeptin-mediated GnRH secretion is sex steroid dependent. Estrogen and progesterone directly modulate kisspeptin activity though the sex-steroid receptors expressed on kisspeptin neurons at both AVPV and the arcuate nucleus (158-160). Furthermore, two distinct populations of kisspeptin neurons, the infundibular/arcuate region of which interacts with neurokinin B and dynorphin, appear to mediate distinct sex-steroid pathways (discussed in more detail in sections 4.1-4.4).

Briefly, in humans, KNDy neurons in the infundibular nucleus alone are involved in negative and positive sex-steroid feedback, whereas in rodents positive sex-steroid feedback seems to be mediated via kisspeptin neurons in the AVPV region and negative sex-steroid feedback via the arcuate KNDy neurons (Figure 2) (67,111,160,161).

Stimulatory Effect of Kisspeptin on GnRH and Gonadotropin Secretion

Kisspeptin is a potent stimulator of the HPG axis – and in fact, it is the most potent GnRH secretagogue currently known. Kisspeptin signals directly to the hypothalamic GnRH neurons via kisspeptin receptor to release GnRH into the portal circulation, which in turn stimulates the anterior pituitary gonadotropes to produce LH and FSH (129,162).

The stimulatory effects of kisspeptin on LH secretion have been documented in animal models (163-166). This is consistent with human studies, where kisspeptin increases both LH and FSH secretion with the preferential stimulatory effect on the former (67,167-177). Kissppetin-54 was first administered in healthy men as an intravenous infusion with dose-dependent rise in LH secretion (169). Since then kisspeptin was administered in different isoforms (kisspeptin-54 and kisspeptin-10), different routes (subcutaneous and intravenous), different types of exposure (continuous and bolus), to healthy men and women and in endocrine disease models with low gonadotropin output, all showing stimulatory effect of kisspeptin on LH secretion (fully reviewed in (67)).

Pulsatile GnRH secretion correlates with LH pulsatility, prompting investigation of the effect of kisspeptin on regulating LH pulse frequency. LH pulse frequency and amplitude were increased following intravenous infusion of kisspeptin-10 in healthy men (172), and subcutaneous bolus of kisspeptin-54 in healthy women (174). The hypothalamic response to kisspeptin-54 and the pituitary response to GnRH are preserved in healthy older men (178). Kisspeptin also stimulates LH pulse frequency in reproductive endocrine disorders of low LH pulsatility, including hypothalamic amenorrhea, defects in the neurokinin B pathway and hypogonadal men with diabetes (96,179,180). Indeed, kisspeptin-54 and kisspeptin-10, as well as kisspeptin agonists like MVT-602 (previously known as TAK-448) are able to stimulate physiological reproductive hormone secretion in individuals with functional hypogonadism related to deficient GnRH secretion, such as in hypothalamic amenorrhea or polycystic ovary syndrome (181,182).

In addition, recent findings have explored further the effects of MVT-602. LH concentration increased in a dose-dependent manner, resembling the amplitude and duration found in the physiological mid-cycle LH surge, proving to be safe and well tolerated throughout the dose range (0.3 – 3.0 mg) (183). This approach to mimicking the physiological response during oocyte maturation and ovulation may have clinical utility for women during medically assisted reproduction.

Kisspeptin regulates GnRH and subsequently gonadotropin secretion through Kiss1r, as suggested by Messager who demonstrated no detectable LH levels in response to kisspeptin in Kiss1r knockout mice (119). The prevention of the stimulatory effect of kisspeptin on LH secretion by GnRH antagonists indicate that kisspeptin action is GnRH-mediated (118,164,184-186). This is further supported by the observation that kisspeptin cause depolarization of GnRH neurons (117) and stimulate GnRH release from hypothalamic explants (187,188). The expression of GnRH mRNA is upregulated in GnRH neurons following kisspeptin administration (189). Moreover, in patients with impaired functional capacity of GnRH neurons (idiopathic hypogonadotropic hypogonadism), the same dose of kisspeptin failed to induce LH response seen in healthy men and women (190). In female rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, confirming its role in the maintenance of normal LH levels and to estrous cyclicity (191).

Some investigators have demonstrated a direct stimulatory effect of kisspeptin on gonadotropes, but this direct stimulatory action of kisspeptin on gonadotropes remains debatable (192-196). Kiss1 and Kiss1r gene expression has been shown in gonadotropes, and gonadotropin secretion from the pituitary explants was observed following exposure to kisspeptin (78,192-195). Moreover, LHβ and FSHβ gene expression was upregulated in the primary pituitary cells treated with kisspeptin. Whilst kisspeptin can directly regulate gonadotropins at the transcriptional level, it appears to be less relevant than the GnRH-mediated action (67,195,196).

Desensitization Effect of Chronic or Continuous Exposure to Kisspeptin

Continuous administration of GnRH desensitizes the HPG axis by downregulation of GnRH receptors and desensitization of gonadotropes, following an initial stimulatory effect (39). It is therefore important to ascertain the effects of continuous exposure to kisspeptin on the HPG axis. Efforts have been made to assess the impact of continuous infusions of kisspeptin in a number of animal experiments (119,197-200).

In adult rats, continuous administration of kisspeptin-54 increased serum LH and free testosterone on day one, but this stimulatory effect was lost after 2 days, indicative of kisspeptin receptor desensitization (200). In rhesus monkeys, the continuous administration of kisspeptin-10 resulted in suppression of LH secretion, indicating desensitization of kisspeptin receptor (198). The kisspeptin receptor has been shown to desensitize in vitro (197). In sheep, infusion of kisspeptin-10 resulted in acute increase in serum LH levels, which declined by the end of 4-hour infusion, while GnRH remained elevated following the discontinuation of kisspeptin-10 administration. This suggests that desensitization to GnRH could be occurring at the level of pituitary gonadotropes (119).

Consistent with animal studies, Jayasena et al. demonstrated that in women with hypothalamic amenorrhea an initial increase in LH and FSH secretion was not sustained following twice daily subcutaneous kisspeptin-54 administration for two weeks (176). Other studies in humans employing continuous or repeated kisspeptin administration provide conflicting evidence for kisspeptin-mediated desensitization and appear to be dose-related (172,180). High doses of kisspeptin may induce desensitization, but this is not apparent at lower doses (67). Sustained LH secretion and increased LH pulsatility was demonstrated with lower dose of kisspeptin-54 (0.01-1nmol/kg/h) infusion for 8 hours in women with hypothalamic amenorrhea (180) and kisspeptin-10 (3.1 nmol/kg/h) infusion for 22.5 hours in healthy men (172). In contrast, LH secretion was not maintained in three healthy men during the 24 hour infusion of kisspeptin-10 at 9.2 nmol/kg/h (the highest dose used in humans so far), although serum LH did not fall to the castrate levels and remained well above baseline at end of infusion (201).

Kisspeptin receptor agonist analogues, TAK-488 and TAK-683, induce desensitization when administered to healthy men (202,203). However, the ability of natural kisspeptin fragments to downregulate the HPG axis in humans remains to be established, and is to date complicated by differences in study protocols, in terms of isoform of kisspeptin used, duration (8 hours-2 weeks), mode and route of kisspeptin administration, lower doses of kisspeptin in human studies compared to animal, and the endocrine profile of the study participants (men versus women versus hypothalamic amenorrhea).

Sexual Dimorphism in Kisspeptin Signaling

The response to kisspeptin is different in men and women. In men, kisspeptin potently stimulates the release of LH, but in women the effect of kisspeptin is variable and dependent on the phase of menstrual cycle (67). Whilst men respond to the modest doses of kisspeptin, LH response to kisspeptin in healthy women is minimal and inconsistent in the early follicular phase but greatest in the pre-ovulatory phase of the menstrual cycle (169-171,177). This indicates that in addition to the fluctuations in sex-steroid milieu, other mechanisms, such as changes in pituitary sensitivity to GnRH or GnRH network responsiveness to kisspeptin regulate the sensitivity to kisspeptin throughout the menstrual cycle (67,126,204).

Not only there is sexual dimorphism in gonadotropin response to kisspeptin, but there are also anatomical differences. Female hypothalami have significantly more kisspeptin fibers and kisspeptin cell bodies than men (173). Only a few kisspeptin cell bodies are present in the male infundibular nucleus and none in the rostral periventricular nucleus, which is on contrary to the female hypothalami with abundant kisspeptin network in both of these hypothalamic nuclei (108). These sex differences in kisspeptin neurons appear to be established early during perinatal development through the action of sex steroids (126,205).

These marked functional and anatomical differences may reflect sexually dimorphic roles of kisspeptin between both sexes, influencing their reproductive functions, namely the sex steroid feedback in GnRH and gonadotropin secretion (67).

Kisspeptin, GnRH and Puberty

Kisspeptin is crucial for normal pubertal development, the discovery of which formed the basis for the obligate role of kisspeptin signaling in the control of reproductive function (206). More than a decade ago two independent groups identified ‘inactivating’ mutations in KISS1R in patients with hypogonadotropic hypogonadism presenting with pubertal delay (85,86). Recently, a male patient with a biallelic loss-of-function KISS1R mutation was described who had undergone a normal and timely puberty, although as a child he had presented with microphallus and bilateral cryptorchidism. This suggests different levels of dependence of the hypothalamic-pituitary-gonadal axis on kisspeptin signaling during the reproductive life span, with the mini-puberty of infancy appearing more dependent on the kisspeptin system than is adolescent puberty (207). On the other hand, activating mutations in KISS1R and KISS1 were then described in children with central precocious puberty (89,90).

Hypothalamic expression of Kiss1 and Kiss1R mRNA is upregulated at puberty (117,165,208), and the percentage of GnRH neurons depolarizing in response to kisspeptin increases from juvenile (25%) to pubertal (50%) and to adult mice (>90%) (117), suggesting that GnRH neurons may acquire sensitivity to kisspeptin across puberty. In monkeys, kisspeptin-54 secretion and pulsatility increased at the onset of puberty (209). Moreover, the exogenous administration of kisspeptin resulted in earlier puberty in rats and monkeys (208,210), whereas kisspeptin antagonists delayed puberty in rats (186) and inhibited GnRH release in pubertal monkeys (211). In other study, daily injections of a synthetic kisspeptin analogue have been shown to significantly advance puberty in prepubertal female mice (212). GnRH neuron-specific Kiss1r knockout mouse showed a delay in pubertal onset, abnormal estrous cyclicity in female and abnormal external genitalia in male (microphallus, decreased anogenital distance associated with failure of preputial gland separation) (213).

Exogenous kisspeptin stimulated GnRH-induced LH secretion in patients with hypogonadotropism resulted in a spontaneous and permanent activation of their hypothalamic-pituitary-gonadal axis, whereas patients with idiopathic hypogonadotropic hypogonadism and no spontaneous LH pulsatility did not respond to kisspeptin, suggesting that the reversal of hypogonadism, sexual maturation and puberty may well be associated with the acquisition of kisspeptin responsiveness which in turn signals the emergence of reproductive endocrine activity (214). A recent study, 15 children with delayed puberty were administered intravenous kisspeptin and displayed divergent responses, with seven subjects having no response to kisspeptin, whereas others having either robust response (comparable to those of adults) or intermediate responses as perceived in one case (215).

GnRH release during puberty appears to require a cooperative mechanism between the kisspeptin/NKB networks in close interaction with different neuropeptides, as substance P, NKA, RFRP-3 and alpha-MSH, working as partners to regulate puberty timing influenced, naturally, by a combination of genetic, environmental, and gene-environment interactions (216).

Agonists and antagonists of kisspeptin and NKB were administered into the stalk-median eminence (region with high concentration of GnRH, kisspeptin and NKB neuroterminal fibers), and it was found that both kisspeptin-10 and the NK3R agonist senktide stimulated GnRH release in a dose-responsive manner in prepubertal and pubertal monkeys. However, senktide-induced GnRH release was blocked in the presence of a KISS1R antagonist and the kisspeptin-induced GnRH release was blocked in the presence of NK3R antagonist in pubertal monkeys, leading to the notion that a reciprocal signaling mechanism between kisspeptin and NKB exists and is possibly necessary for a normal puberty (217). These data together emphasizes that disrupted kisspeptin-GPR54-NKB signaling leads to hypogonadotropic hypogonadism, reinforcing the critical role of kisspeptin in puberty.

REGULATION OF GnRH AND GONADOTROPIN SECRETION

Development and maintenance of normal reproductive function requires a coordinated interplay between neuroendocrine, metabolic, and environmental factors. The GnRH-gonadotropin system plays a central role in the regulation of reproduction by integrating different signals and factors (Figure 3) (126,204).

Figure 3. Neuroendocrine regulation of GnRH/gonadotropin secretion.
The GnRH-gonadotropin system plays a central role in the regulation of reproduction by integrating different neuroendocrine, metabolic and environmental signals/factors. The KNDy signaling has a key role in this process by integrating some of these signals and by regulating GnRH neurons.

Overview of Sex Steroid Feedback

A crucial role for sex steroids in the regulation of GnRH neurons and/or gonadotropes in humans was initially proposed as serial blood sampling and gonadotropin assays in women through phases of menstrual cycle showed an uneven distribution, with a clear mid-cycle surge in LH and FSH. Two mechanisms were proposed to mediate this effect: first, GnRH secretion is altered in response to the steroid milieu; second, sensitivity of the gonadotropes to a GnRH input is sex-steroid dependent, although the exact mechanism remains controversial due to inter-species variation (218).

Hypothalamic secretion of GnRH increases during proesterus in rats (219), sheep (220), and non-human primates (221). Pulsatile once hourly administration of exogenous GnRH restored ovulation in Rhesus monkeys with hypothalamic lesions which abolished GnRH secretion, suggesting that it was the ‘ebb and flow’ of ovarian estrogen feedback acting directly on the pituitary which triggered an LH surge (222). In humans, endogenous GnRH secretion is potentially diminished during the pre-ovulatory LH surge and the suppression of gonadotropin secretion is greater with lower doses of a GnRH receptor antagonist during the mid-cycle surge in comparison to the other phases of the menstrual cycle (223). This suggests that pituitary gonadotrope sensitivity to GnRH is enhanced during the mid-cycle surge. Administration of exogenous estradiol or testosterone in men with hypogonadotropic hypogonadism receiving pulsatile GnRH therapy, decreased gonadotropin concentrations, demonstrating inhibitory effects of sex-steroids at the level of pituitary (224). A direct effect of estrogen on gonadotropes is further demonstrated by the inhibition of LH secretion from rat pituitary gonadotropes in vitro (225). Literature to date suggests that there is dual-site sex-steroid feedback in the regulation of gonadotropin secretion, occurring at the level of both pituitary and hypothalamus (226-231).

Estrogen Feedback

Patterns of GnRH and LH secretion across the menstrual cycle are modulated by estradiol feedback. A biphasic effect of estradiol on gonadotropin secretion has long been established and it is essential for normal menstrual cycle, with an initial negative feedback (greater suppression of FSH) and a subsequent positive feedback (more prominent for LH) (32). However, the basis for estrogen feedback has been unclear for a long time. GnRH neurons do not express estrogen receptor alpha (ER-alpha) (232,233), and therefore a mediator between gonads and hypothalamus was missed. Recent evidence suggests that kisspeptin and neurokinin B (132) appears to be providing this “missing link” as a key regulator of both negative and positive estrogen feedback (67,126).

KNDy neurons in the infundibular nucleus in humans and the arcuate nucleus in other mammals mediate negative estrogen feedback. Estrogen suppresses kisspeptin and neurokinin B release from KNDy neurons, which reduce their stimulatory input to GnRH neurons. Simultaneously, there is a relative deficiency in dynorphin signaling as part of this negative feedback, releasing the inhibitory action on kisspeptin signaling (Figure 2) (67). Immunohistochemical staining of the postmenopausal women hypothalami showed up-regulated expression of KISS1 mRNA and hypertrophy of kisspeptin neurons in the infundibular nucleus when compared to the premenopausal women (111). These hypertrophied kisspeptin neurons co-localized with ER-alpha, had increased expression of neurokinin B and decreased levels of prodynorphin mRNA (234-236). The above evidence for the involvement of the infundibular KNDy system in mediating negative estrogen feedback in humans is consistent with animal studies. Kisspeptin neurons in the arcuate nucleus show frequent co-localization with ER-alpha (160,237). In ovariectomized animals, the expression of Kiss1 and neurokinin B mRNA was up-regulated but prodynorphin mRNA reduced in the arcuate nucleus (equivalent to the infundibular nucleus in humans), and this was reversed by estrogen replacement (102,115,120,238-242). Postmenopausal women are resistant to the stimulatory effect of kisspeptin on LH secretion (142,243), but postmenopausal women receiving estradiol replacement therapy are only resistant to kisspeptin initially and then they do demonstrate a remarkable increase in LH pulse amplitude with direct correlation to the circulating levels of estradiol and duration of kisspeptin administration (243). However, neurokinin B regulates gonadotropin secretion in postmenopausal women, and antagonizing the neurokinin 3 receptor modestly decreases LH secretion in this context (142).

Interestingly, the use of neurokinin 3 receptor antagonists has been shown to effectively reduce the frequency and severity of menopause-related vasomotor symptoms owing to their inhibitory effect in the hypothalamic thermoregulatory center, and thus presenting a potential non-hormonal treatment option for menopausal women (144,148,149).

Negative estrogen feedback switches to positive feedback in the late follicular phase of menstrual cycle, in order to induce the pre-ovulatory LH surge. Ovarian estradiol seems to be the predominant signal to trigger this switch, via ER-alpha, stimulating RP3V kisspeptin neurons while it inhibits arcuate kisspeptin neurons. Recent evidence supports the role of kisspeptin in generating the LH surge: during an assisted conception cycle, kisspeptin-54, used instead of a routinely administered human chorionic gonadotropin, induced an LH surge, and oocyte maturation, with a subsequent live term birth (241). Repeated twice-daily administration of kisspeptin-54 shortened the menstrual cycle, suggesting that the onset of LH surge was advanced (173). This is further supported by antagonistic studies in animal models, where the administration of kisspeptin antiserum or antagonists blunt/prevent LH peak, whilst kisspeptin advances LH surge (211,244,245).

However, kisspeptin-mediated positive estrogen feedback has marked anatomical variations between humans and other species. In rodents, positive estrogen feedback is mediated via the AVPV nucleus, which is absent in humans, other primates and sheep (Figure 2). AVPV neurons are sexually dimorphic, with higher density of ER-alpha described in females and AVPV kisspeptin neurons, as a subset of AVPV neurons, share this pattern (246). There seems to be functional specialization, since only a subset of AVPV kisspeptin neurons (~1/3) are synaptically connected to GnRH cell bodies, but of these, nearly all express estrogen sensitivity and most co-express tyrosine hydroxylase to facilitate positive feedback (247). The expression of Kiss1 mRNA in the AVPV nucleus is low following an ovariectomy but is dramatically increased with estrogen treatment and at the time of LH surge (160,161). In sheep, positive estrogen feedback is mediated though the arcuate nucleus, where the expression of Kiss1 mRNA is the greatest at the pre-ovulatory LH surge (195).

There are no studies looking at the anatomical region of estrogen mediating positive feedback in humans. Although there does not appear to be two distinct anatomical populations of kisspeptin neurons to relay negative and positive sex-steroid feedback in humans, it is possible that separate signaling pathways exists to mediate gonadal steroid feedback.

Whilst it is clear that kisspeptin is involved in estrogen-induced mid-cycle gonadotropin surge, the role of KNDy neurons in positive estrogen feedback is less obvious. In sheep, the expression of neurokinin B mRNA was increased during the LH surge, and neurokinin B receptor agonist senktide induced LH secretion mimicking its mid-cycle surge (248,249). However, this has not been reproduced in other species, including humans (180). In summary, KNDy neurons mediate negative estrogen feedback in the infundibular nucleus in humans and the arcuate nucleus in other species. Positive estrogen feedback is mediated via kisspeptin neurons, which show marked inter-species anatomical variation.

In addition to the gonads, the brain is one of the major organs producing estradiol, and recently a number of studies demonstrated that estradiol is synthesized and released in the hypothalamus (i.e. neuroestradiol) contributing to the regulation of GnRH release, particularly regarding its positive feedback effect during the preovulatory GnRH/LH surge (250).

Progesterone Feedback

Progesterone reduces LH pulse frequency in healthy women. LH secretory pattern in women exposed to exogenous progesterone was comparable to LH profile observed in the mid-luteal phase, demonstrating that progesterone plays a central role in the luteal phase of menstrual cycle (251). These inhibitory effects of progesterone on gonadotropin secretion are mediated by the progesterone receptor (PR) (252). The suppressive effect of progesterone on LH secretion was diminished in the context of estrogen deficiency, while co-administration of estradiol restored it (252), suggesting an interplay between these sex steroids. However, the presence of PR on only a small subset of GnRH neurons (253-255)led to the notion that intermediaries are involved in mediating inhibitory progesterone signal to GnRH neurons.

There is evidence that KNDy neurons play a role in mediating progesterone feedback on GnRH through dynorphin signaling (Figure 2) (102,120). PR have been demonstrated to be co-localized with dynorphin in the KNDy neurons (159)and progesterone increased dynorphin concentrations (256). Moreover, the number of preprodynorphin mRNA expressing cells decreased in postmenopausal women (236) and in ovariectomized ewes, but normalized with exogenous progesterone to luteal levels (256).

Testosterone Feedback

Testosterone exerts negative feedback on gonadotropin secretion. Early studies verified that LH and FSH pulse frequency are enhanced in hypogonadal men and exogenous testosterone decreases gonadotropin secretion, suggesting that testosterone have an inhibitory effect on GnRH secretion (230,257).

Few GnRH neurons express androgen receptors (AR) (258). GnRH neurons were thus considered to be reliant on an intermediary neuronal population to mediate testosterone feedback. A key role for KNDy neurons in this mediation has been suggested, as these neurons express AR which directly mediate the androgen feedback. The androgen feedback may also rely on the aromatization of testosterone, as testosterone-induced suppression of Kiss1 mRNA in the rodent arcuate nucleus is identical to that observed with estradiol, but more than that observed with dihydrotestosterone administration (259). The cross-talk between AR and ER was suggested from animal studies: AR expression was downregulated in the prostate following neonatal estrogen exposure (260), and AR transcription was modulated following a co-transfection of AR and ER (261).

Navarro has described a role for KNDy neurons in mediating the negative testosterone feedback on GnRH secretion, and provided evidence that neurokinin B released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in male mice: Kiss1 and dynorphin mRNA are regulated by testosterone through estrogen and androgen receptor-dependent pathways; KNDy neurons express neurokinin B receptor whereas GnRH neurons do not, and senktide (an agonist for the neurokinin B receptor) activates KNDy neurons leading to gonadotropin secretion but has no discernible effect on GnRH neurons (262). Other studies demonstrated that the suppression of gonadotropin secretion using testosterone is associated with a reduction of Kiss1 mRNA in the hypothalamus (118,208,263). Moreover, post-orchidectomy rise in LH in rodents can be blocked by kisspeptin antagonists, further suggesting that kisspeptin system mediates the hypothalamic androgen feedback (186).

Stress and Glucocorticoids

Physical and psychological stress is associated with hypothalamic amenorrhea, possibly though the activation of hypothalamic-pituitary-adrenal (HPA) axis (264,265). Experimental evidence points towards a cortisol-mediated suppression of gonadotropin secretion as the main key pathway to explain stress-induced gonadotropin suppression(55,266-273). The negative effect of cortisol on HPG axis is recognized to occur at both pituitary and hypothalamic levels. There are also data suggesting that upstream factors in the HPA axis, such as Corticotropin Releasing Hormone (CRH) and vasopressin may play a mediatory role (274,275).

Cortisol secretion in women with hypothalamic amenorrhea is elevated (267), and evening adrenocorticotropic hormone (ACTH) and cortisol concentrations are higher in excessive exercise (266,270). Administration of exogenous glucocorticoids to eugonadal women was associated with a decrease in LH pulse frequency, suggesting that glucocorticoids have a negative action on GnRH secretion (273). In ovine portal blood, cortisol administration led to a decrease in GnRH pulse frequency (272). Inferences of cortisol effects on gonadotropin secretion were also derived from observations in women and men with Cushing’s syndrome (condition associated with excessive cortisol secretion), where exogenous GnRH preferentially stimulates FSH whilst LH remains unchanged (268,271). The resolution of male hypogonadotropic hypogonadism was also observed in men with remission of Cushing’s disease (271). This negative input of cortisol on the HPG axis may be modulated by sex-steroid hormones, and kisspeptin signaling has also been implicated in the process.

Cortisol alone had no impact on GnRH pulsatility in ovariectomized ewes, but the co-administration of estradiol and progesterone led to a 70% decrease in GnRH secretion (272). Decreased hypothalamic Kiss1 mRNA expression has been observed during exposure to stress or exogenous glucocorticoids. The role of kisspeptin in mediating stress inputs is further supported by the expression of glucocorticoid receptor on murine kisspeptin neurons (276). Colocalization by immunohistochemistry of CRH receptor (CRH-R) in most hypothalamic kisspeptin neurons in the AVPV/PeN and ARC nuclei as well as glucocorticoid receptor (GR) in AVPV/PeN kisspeptin neurons support a relevant direct role of kisspeptin neurons in the inhibitory effects of CRH/ glucocorticoids (277).

Hypothalamic CRH neurons, important regulators of the stress response, also directly modulate GnRH excitability in a dose-dependent and receptor-specific manner, and the GnRH response to CRH is influenced by estrogens (278). Intracerebroventricular administration of CRH in female rats suppressed LH pulsatility and the LH surge, and this suppression was enhanced by estrogens (279).

Animal models have also linked increased exposure to RFamide-related peptide-3 (RFRP-3) during acute and chronic stress and hypothalamic expression of GnIH mRNA. Along these lines, the surface of GnIH neurons has glucocorticoid receptors and hydrocortisone administration was associated to an increased GnIH mRNA expression, ultimately leading to lower GnRH activity and dysregulation of the HPG axis (280). Together, these findings emphasize that kisspeptin as GnIH provide relevant inputs that contribute to an inhibitory effect of corticosteroids on gonadal axis during stress.

Prolactin

Prolactin is a well-known inhibitor of GnRH release and a suppressor of the HPG axis. The association between hyperprolactinemia and reproductive dysfunction has long been established, accounting for 14% of secondary amenorrhea and hypogonadism cases (281) and for a third of women presenting with infertility (282,283). Hyperprolactinemia is evident in 16% of men with erectile dysfunction and in 11% of men with oligospermia (284). The decreased pulsatility of LH in hyperprolactinemia responds to bromocriptine (285). GnRH therapy has restored ovulation and normal luteal function in bromocriptine resistant hyperprolactinemia women (286,287), suggesting that prolactin exerts inhibition through direct reduction of GnRH secretion.

The neuroendocrine pathway by which prolactin inhibits GnRH pulse frequency remains to be fully elucidated. A direct action of prolactin on the GnRH neuronal network is possible (288,289). Prolactin has also been demonstrated to influence other systems, including GABA (290), β endorphins (291), neuropeptide Y (292) and dopaminergic systems (via tuberoinfundibular dopamine (TIDA) neurons) (293).

Nevertheless, data suggest that prolactin receptors are expressed in most kisspeptin neurons but only in a small proportion of GnRH neurons, indicating that kisspeptin signaling may have a role in this context (288,294). In rodent models, kisspeptin neurons in the arcuate nucleus modulate dopamine release from dopaminergic neurons, thereby regulating prolactin secretion (295). Kiss1 expression is decreased in lactation, a physiological state associated with hyperprolactinemia (296). Prolactin-sensitive GABA and kisspeptin neurons were identified in regions of the rat hypothalamus (294). Moreover, in a mouse model of anovulatory hyperprolactinemia (induced by a continuous infusion of prolactin), Kiss1 mRNA levels were diminished and peripheral administration of kisspeptin restored gonadotropin secretion and ovarian cyclicity (297). There are also other animal studies reporting an inhibitory effect of prolactin on Kiss1 expression (298,299). This data suggests that kisspeptin is a possible link between hyperprolactinemia and GnRH deficiency. The administration of kisspeptin-10 reactivated the gonadotropin secretion in women with hyperprolactinemia-induced hypogonadotropic amenorrhea, suggesting that GnRH deficiency in the context of hyperprolactinemia is, at least in part, mediated by an impaired hypothalamic kisspeptin secretion (300).

On the other hand, kisspeptins appears to have a stimulatory effect on prolactin release, as demonstrated in a recent study in ovariectomized rats which had intracerebroventricular injections of kisspeptin-10 with subsequent increase in prolactin release, and this required the estrogen receptor-alpha and was potentiated by progesterone via progesterone receptor activation (301).

Nutrition and Metabolism

A link between energy balance and reproductive function enables organisms to survive to reproductive maturity and to withstand the energy needs of parturition, lactation, and other parental behaviors. This link optimizes reproductive success under fluctuating metabolic conditions (302). Kisspeptin signaling may link nutrition/metabolic status and reproduction by sensing energy stores and translate this information into GnRH secretion (303). These relations elucidate further associations between reproductive dysfunction and metabolic disturbances, such as diabetes, obesity or anorexia nervosa (67,304,305).

Food deprivation impairs GnRH and gonadotropin secretion, and leptin (a satiety hormone secreted by adipose tissue, the levels of which drop in response to fasting) plays a role in this inter-regulation by stimulating LH release (67,306-308). Periods of fasting and calorie restriction decrease LH pulse frequency and increase pulse amplitude (302,309-311). Administration of recombinant leptin increased LH pulse frequency in women with hypothalamic amenorrhea (312) and prevented fasting-induced drop in testosterone and LH pulsatility in healthy men (313). Moreover, humans with mutations in leptin or in leptin receptor show hypogonadism (314). Thus, the crosstalk between kisspeptin and leptin is relevant for reproduction and fertility (71), including in the setting of assisted reproduction techniques (315).

Kisspeptin neurons may have a role in mediating the metabolic signals of leptin on the control of HPG axis, as 40% of the arcuate kisspeptin neurons express leptin receptors in contrast to the GnRH neurons, where leptin receptors are absent (316-319). Food deprivation is associated with a decrease in kisspeptin, and subsequent reduction in gonadotropin secretion (320-323). Levels of low Kiss1 mRNA expression in the leptin-deficient ob/ob mice are partially upregulated by exogenous leptin (161). Moreover, exogenous kisspeptin restored vaginal opening (marker of sexual maturation) in malnourished rodents (320). Animal models of type 1 diabetes, characterized by insulin deficiency and impaired cellular nutrition, had hypogonadotropic hypogonadism and decreased Kiss1 mRNA expression. Repeated administration of kisspeptin to these rodents increased prostate and testis weight (324). It is plausible that a relative deficiency of kisspeptin secretion is a mechanism for hypogonadotropic hypogonadism in patients with obesity and diabetes (179). In hypogonadal men with type 2 diabetes, kisspeptin-10 increased LH secretion and pulse frequency (179). Although early studies appeared to suggest a direct link between kisspeptin and leptin, it seems that the neuronal pathway whereby leptin modulates GnRH is far more complex (325,326). Only partial restoration in Kiss1 mRNA in leptin-deficiency and normal pubertal development and fertility observed in selective leptin receptor deletion from kisspeptin neurons suggest that kisspeptin may link reproduction and metabolism through other ways than leptin (161,327). Proopiomelanocortin (POMC), agouti-related peptide, neuropeptide Y, ghrelin, and cocaine- and amphetamine-regulated transcript (CART) expressing neurons have been linked to this process (303,319). Kisspeptin neurons communicate with POMC and neuropeptide Y neurons and are able to modulate the expression of relevant genes in these cells (316). This link between kisspeptin and other peptides classically associated to food intake (as POMC and neuropeptide Y) was explored due to the anorexigenic effect of intracerebroventricular administration of KP-10 in male rats mediated via anorectic neuropeptides, nesfatin-1 and oxytocin, expressed in various hypothalamic nuclei. Diminished food intake and anorexia was significantly abolished by pretreatment with oxytocin receptor antagonist (328,329).

Several studies have also suggested that ghrelin can interact directly with hypothalamic neurons leading to suppression of gonadotropins release, and thus impairing fertility, an effect that is dependent of the estradiol milieu (303,330-332).

GABA (Gamma-Amino Butyric Acid)

GABA has also been implicated as a regulator of GnRH secretion. Although GABA is classically an inhibitory neurotransmitter in the central nervous system, most mature GnRH neurons are stimulated by GABA, which has attributed to GABA an excitatory action in HPG axis. The precise physiology of this mechanism is still unclear (333-337), but it may be related to the bidirectional interactions between GABA and kisspeptin pathways, as well as between these and GnRH neurons, in a variety of ways throughout development (338). In early development, GABA seems to increase KISS1 expression in embryonic phase and early postnatally, while in the absence of GABAergic input the expression of KISS1 declines (338,339). In the prepubertal period, the central restraint on GnRH secretion seems to be mediated by GABA possibly acting directly via kisspeptin neurons (338). In the peri-pubertal phase, the antagonism of GABA and the intrinsic disinhibition of kisspeptin neurons seem to be critical in puberty initiation and development (340,341). In adulthood, the interactions between GnRH-GABA-kisspeptin become more complex with HPG axis function critically dependent on such interactions. For instance, the preovulatory surge does not occur in the absence of GABA signaling, thus neurons co-expressing GABA and kisspeptin seem crucial in providing double excitatory input to GnRH neurons at the time of ovulation (338,342).

Additionally, in healthy men, total endogenous GABA levels in the anterior cingulate cortex, a key limbic structure, significantly decreased after intravenous infusion of kisspeptin (1 nmol/kg/h) demonstrating a potent inhibitory effect of kisspeptin on GABA levels which could be a fundamental concept in understanding the central limbic effect of kisspeptin in the human brain (343).

Other Neuropeptides

In addition to KNDy system and GABA, other peptides and neurotransmitters have been shown to influence GnRH-gonadotrope system: vasoactive intestinal polypeptide (VIP), vasopressin, catecholamines, nitric oxide, neurotensin, gonadotropin-inhibitory hormone (GnIH) /RFamide related peptide-3 (RFRP-3) (337), nucleobindin-2/nesfatin-1 (344). Excitatory inputs to the HPG axis may be mediated by VIP, catecholamines, glutamate and possibly vasopressin, whereas GnIH in birds, or its mammalian homolog RFRP-3, provide inhibitory inputs (345-349). RFRP neuronal populations have been detected mainly in the hypothalamic dorsomedial nucleus or adjacent regions, and they have projections to several hypothalamic areas including the arcuate nucleus, paraventricular nucleus, ventromedial nucleus and the lateral hypothalamus, all areas with major roles in the regulation of reproduction and energy balance (350,351). RFRP-3, encoded by the gene Rfrp, inhibits the electric firing of GnRH and kisspeptin neurons (346,352), which results in a suppression of GnRH-induced gonadotropin release with consequent inhibition of the reproductive axis (353). This RFRP-3 inhibitory input on the gonadotropin release is influenced by estrogens and may well be involved in their negative feedback. Estrogens reduce RFRP-3 expression and RFRP-3 neuronal activation (354,355).

Particular attention has been paid to the role of glutamate as a stimulatory modulator of the activity of ARC kisspeptin neurons, reaffirming the role of kisspeptin as a major neural integrator of inputs to GnRH neurons. Data from Kiss1 KO rats showed failure to increase GnRH/LH secretion following monosodium glutamate/NMDA administration (356).

SUMMARY

Complex neuroendocrine networks coordinate the regulation of reproduction, integrating a wide range of internal and external environmental inputs and signals. GnRH, the principal regulator of reproduction integrates cues from sex steroids, stress, glucocorticoids, nutritional and metabolic status, prolactin and other peptides, to controls gonadotropin secretion and subsequently gonadal function. Recently, the KNDy neuronal network has emerged as essential gatekeeper of GnRH release and thus reproduction, fertility and puberty. Translational clinical studies, exploring kisspeptin and neurokinin B activity in various physiological and pathological states are pivotal to explore potential clinical applications for these novel neuropeptides and their agonists as well as antagonists, may underpin future management of some disorders with dysfunctional GnRH pulsatility, such polycystic ovary syndrome, hypothalamic amenorrhea, infertility, obesity, pubertal disorders and menopause-related symptoms.

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Calcium & Phosphate Metabolism and Related Disorders During Pregnancy and Lactation

ABSTRACT

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

INTRODUCTION

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

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

MINERAL PHYSIOLOGY DURING PREGNANCY

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

Mineral Ions

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

Figure 2. Schematic depiction of longitudinal changes in calcium, phosphorus, and calciotropic hormone levels during human pregnancy. Shaded regions depict the approximate normal ranges. PTH does not decline in women with low calcium or high phytate intakes and may even rise above normal. Calcidiol (25OHD) values are not depicted; most longitudinal studies indicate that the levels are unchanged by pregnancy but may vary due to seasonal variation in sunlight exposure and changes in vitamin D intake. FGF23 values cannot be plotted due to paucity of data. Reproduced with permission from (1).

Parathyroid Hormone

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

Vitamin D Metabolites

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

Total calcitriol levels increase two to five-fold early in pregnancy and stay elevated until parturition, whereas measured free calcitriol levels were reported to be increased only in the third trimester (12). However, when the 20-40% increase in vitamin D binding protein and the decline in serum albumin during pregnancy are considered, the calculated free calcitriol should be increased in all three trimesters (11,13-16).There are several unusual aspects about this situation. PTH is normally the main stimulator of the renal 1α-hydroxylase (CYP27B1); consequently, elevated calcitriol values are usually driven by high PTH concentrations. An exception to this is the ectopic expression of an autonomously functioning 1α-hydroxylase by such conditions as sarcoidosis and other granulomatous diseases. Another exception is pregnancy because the rise in calcitriol occurs when PTH levels are typically falling or quite low. Moreover, this increase in calcitriol occurs despite the ability of high levels of fibroblast growth factor-23 (FGF23) to suppress the synthesis and increase the catabolism of calcitriol, as shown in animal models of X-linked hypophosphatemic rickets (17-19). Evidence from additional animal models suggest that it is not PTH but other factors, such as PTH-related protein (PTHrP), estradiol, prolactin, and placental lactogen, which drive the 1α-hydroxylase to synthesize calcitriol (1).

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

Calcitonin

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

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

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

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

Fibroblast Growth Factor-23 (FGF23)

Intact FGF23 doubles its concentration in the mother’s circulation during rodent pregnancies (17-19), but whether it increases during human pregnancy is not clear. A small longitudinal study of 12 women found that intact FGF23 doubled in the third trimester over values in the first and second trimesters (33), whereas a larger longitudinal study of 81 women found no difference in intact FGF23 values at 36 weeks of gestation and 3-6 months post-weaning (34). Within 24 hours after delivery in another study, mean intact FGF23 did not differ between postpartum women and non-pregnant women (35).

Other Hormones

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

Estradiol increases to about 100-fold the levels obtained during normal menstrual cycles and may be influencing bone metabolism. As noted earlier, estradiol has been postulated to be one of the stimulators of the 1α-hydroxylase, which synthesizes high levels of calcitriol during normal pregnancy, and even in the absence of PTH (1).

Prolactin and placental lactogen both increase during pregnancy and activate prolactin receptors. Osteoblasts express prolactin receptors, and prolactin receptor deficient mice show decreased bone formation (36). Suppressing the prolactin level with bromocriptine blunted a pregnancy-related gain in bone mineral content in rats (37). These data are consistent with the notion that prolactin or placental lactogen regulate skeletal metabolism during pregnancy. Furthermore, prolactin can indirectly affect skeletal metabolism by stimulating PTHrP synthesis and release from the breasts (38-40).

Circulating oxytocin levels also rise during pregnancy (41), and the oxytocin receptor is expressed by osteoclasts and osteoblasts (42). Male and female mice lacking oxytocin or its receptor have an osteoporotic phenotype with low bone formation (43). Oxytocin has been shown to stimulate osteoblast differentiation and function, stimulate osteoclast formation, but inhibit osteoclast function and skeletal resorption (43,44). Taken together, these data predict that oxytocin may regulate bone metabolism during pregnancy, but this has not been directly studied in vivo.

Intestinal Calcium and Phosphate Absorption

Intestinal absorption of calcium doubles as early as 12 weeks of human pregnancy, as shown by clinical studies that used stable isotopes of calcium, and by other calcium balance studies (1). This increase in calcium absorption appears to be the major maternal adaptation to meet the fetal need for calcium. It has been generally believed that the doubling or tripling of calcitriol levels explains the increased intestinal calcium absorption and concurrent increases in the intestinal expression of calbindin_9k-D (S100G), TRPV6, Ca²⁺-ATPase (PMCA1), and other genes and proteins involved in calcium transport. However, intestinal calcium absorption doubles in the first trimester, well before the rise in free calcitriol levels during the third trimester. Animal studies have indicated that placental lactogen, prolactin, and other factors may stimulate intestinal calcium absorption (1) and that calcitriol or the vitamin D receptor are not required for intestinal calcium absorption to increase during pregnancy (1,23,45-48).

The peak fetal demand for calcium does not occur until the third trimester, and so it is unclear why intestinal calcium absorption should be upregulated in the first trimester. It may allow the maternal skeleton to store calcium in advance of the peak demands for calcium that occur later in pregnancy and lactation; some studies in rodents have shown this to be the case with the bone mineral content rising significantly before term (17,26,47). Women have also been found to be in a positive calcium balance by mid-pregnancy (49), likely due to the effect of increased intestinal calcium absorption on skeletal mineralization.

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

Renal Handling of Calcium

The doubling of intestinal calcium absorption in the first trimester means that the extra calcium must be passed to the fetus, deposited in the maternal skeleton, or excreted in the urine. Renal calcium excretion is increased as early as the 12th week of gestation, and 24-hour urine values (corrected for creatinine excretion) often exceed the normal range. Conversely, fasting urine calcium values are normal or low, confirming that this hypercalciuria is a consequence of the enhanced intestinal calcium absorption (1). This is absorptive hypercalciuria and will not be reliably detected by spot or fasting urine samples that have been corrected for creatinine concentration. Absorptive hypercalciuria contributes to the increased risk of kidney stones during pregnancy. That women commonly develop hypercalciuria in pregnancy is an indication that they normally absorb more calcium than needed by the fetus, provided that their calcium intake is not low, or that the gastrointestinal absorption of calcium is not impaired by high phytate consumption or malabsorptive disorders.

This absorptive hypercalciuria also renders nomograms of fractional calcium excretion invalid for the diagnosis of familial hypocalciuric hypercalcemia during pregnancy (50,51).

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

Hypocalciuria during pregnancy has been associated with pre-eclampsia, pregnancy-induced hypertension, and low (equal to non-pregnant values) serum calcitriol (52-55). These changes appear largely secondary to disturbed renal function and reduced creatinine clearance, rather than being causes of the hypertension. However, calcium supplementation reduces the risk of pre-eclampsia in women within the lowest quintile of calcium intake (see section J. Low and High Calcium Intake, below), and so there is a pathophysiological link between calcium metabolism and pregnancy-induced hypertension (1).

Skeletal Calcium Metabolism and Bone Density/Bone Marker Changes

As mentioned earlier, some studies in rodents indicate that bone mineral content increases during pregnancy, and other studies have shown that histomorphometric parameters of bone turnover are increased at this time. Systematic studies of bone histomorphometry from pregnant women have not been done. However, one study of 15 women who electively terminated a pregnancy at 8-10 weeks found bone biopsy evidence of increased bone resorption, including increased resorption surface and increased numbers of resorption cavities (56). These findings were not present in biopsies obtained from 13 women at term, or in the non-pregnant controls. This study bears repeating but it does suggest that early pregnancy induces skeletal resorption.

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

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

Another way of assessing whether the maternal skeleton contributes to calcium regulation during pregnancy is to measure bone mineral content or density. A few sequential areal bone density (aBMD) studies have been done using older techniques (single and/or dual-photon absorptiometry, i.e., SPA and DPA), and none with newer techniques (DXA or qCT) due to concerns about fetal radiation exposure. Studies of aBMD are known to be confounded by changes in body composition, weight and skeletal volumes, and all three of these factors change during normal pregnancy. The longitudinal studies used SPA or DPA and found no significant change in cortical or trabecular aBMD during pregnancy (1). Most recent studies examined 16 or fewer subjects with DXA prior to planned pregnancy (range 1-18 months prior, but not always stated) and after delivery (range 1-6 weeks postpartum) [studies reviewed in detail in (57)]. One study found no change in lumbar spine aBMD measurements obtained pre-conception and within 1-2 weeks post-delivery, whereas the other studies reported 4-5% decreases in lumbar aBMD with the postpartum measurement taken between 1-6 weeks post-delivery. A larger study from Denmark obtained DXA measurements of hip, spine, and radius at baseline (up to 8 months before pregnancy) and again within 15 days of delivery in 73 women (58). DXA of the radius was also obtained once each trimester. aBMD decreased between pre-pregnancy and post-pregnancy by 1.8% at the lumbar spine, 3.2% at the total hip, 2.4% at the whole body, 4% at the ultradistal forearm, and 1% at the total forearm, whereas it increased by 0.5% at the proximal 1/3 forearm (58). All women went on to breastfeed, which means that the final aBMD values were confounded by lactation-induced bone loss (see lactation section). These changes in aBMD were statistically significant when compared to 57 non-pregnant controls who also had serial measurements done, but the magnitudes of change were small, and would not be considered statistically significant for an individual woman.

Ultrasound measurements of the os calcis and fingers have been examined in other longitudinal studies, which reported a progressive decrease in indices that correlate with volumetric BMD (1,57). Whether observed changes in the os calcis accurately indicate a true or clinically meaningful decrease in volumetric BMD or imply that losses of BMD are occurring in the spine or hip during pregnancy, is not known. The reliability or relevance of data obtained from ultrasound is questionable since this technique failed to detect any change in volumetric BMD at the os calcis during lactation (59), even though substantial bone loss occurs at the spine and hip during lactation (see lactation section).

Overall, the existing studies have insufficient power to allow a firm conclusion as to the extent of bone loss that might occur during pregnancy, but it seems likely (especially when data from the Danish study are considered) that modest bone loss occurs, which would be difficult to discern on an individual basis. In the long term, pregnancy does not impair skeletal strength or lead to reduced bone density. Several dozen epidemiological studies of osteoporotic and osteopenic women have failed to find a significant association of parity with bone density or fracture risk (1,60), and many have shown a protective effect of parity (61-78).

DISORDERS OF CALCIUM AND BONE METABOLISM DURING PREGNANCY

Osteoporosis in Pregnancy (and Especially Lactation)

In much of the following, the discussion encompasses osteoporosis that may present in pregnancy, the puerperium, or during lactation, so called pregnancy and lactation-associated osteoporosis (PLO). There can be a continuum of changes in bone metabolism from pre-pregnancy, during pregnancy, in the puerperium, and into the breastfeeding and post-weaning intervals. Most (80-90%) of fractures occur in women during lactation, which indicates that the changes during lactation can be more critical than those that happen during pregnancy. For simplicity, most of the discussion occurs in this section, with a shorter discussion in the lactation section of this chapter. This is also necessary because much of the literature does not distinguish between osteoporosis of pregnancy versus lactation, and because osteoporosis presenting in lactation may have been caused in part by bone loss that occurred during pregnancy.

Rarely woman will present with a fragility fracture (most commonly a vertebral fracture, but appendicular fractures also occur) during the third trimester or puerperium, and especially during lactation. Low bone mineral density is usually then confirmed on a subsequent DXA (79). In most cases an aBMD value prior to pregnancy is not available because it was never indicated to be done in women who were until then thought to be healthy. Therefore, the extent of bone loss that occurred during pregnancy or lactation is unknown in most cases, and it is not possible to exclude that low bone density or skeletal fragility preceded pregnancy. In favor of a genetic predisposition is the report that among 35 women who presented with pregnancy associated osteoporosis, there was a higher than expected prevalence of fragility fractures in their mothers (80). A positive family history of osteoporosis has been found in about one-third of patients presenting with vertebral fractures in association with pregnancy or lactation (81-83). Whole genome screening has been done in other case series with pathogenic mutations found in 25-30% of women with PLO and involving such genes as COL1A1, LRP5, and WNT (79,84). Women with genetic mutations tended to have a more severe PLO, as indicated by lower aBMD or a higher number of fractures (82).

It is conceivable that pregnancy may induce significant skeletal losses in some women and, thereby, predispose to fracture. The normal pregnancy-induced changes in mineral metabolism may cause excessive resorption of the skeleton in selected cases, and other factors such as low dietary calcium intake and vitamin D insufficiency may contribute to skeletal losses (79). If calcium intake is very low or a malabsorptive disorder is present, skeletal resorption must occur to maintain the calcium supply to the fetus and placenta. A high rate of bone turnover is an independent risk factor for fragility fractures outside of pregnancy, and so the apparently increased bone resorption observed during pregnancy may increase fracture risk. In favor of pregnancy inducing fragility through excess skeletal losses is an observational study of 13 women with pregnancy-associated osteoporosis who were followed for up to eight years. Since the bone mineral density at the spine and hip increased significantly during follow-up in these women, the investigators concluded that significant bone loss must have occurred during the pregnancy (85). Other case series have documented spontaneous 10-20% increases in bone density in women after they fractured during pregnancy or the puerperium (1,86). Taken together, fragility fractures in pregnancy or the puerperium may result from the combination of abnormal skeletal microarchitecture or fragility preceding pregnancy, and increased bone resorption that occurred during pregnancy. In other words, the woman may have entered pregnancy with a normal skeleton that then experienced excessive resorption. Alternatively, she may have had lower bone density and bone strength prior to pregnancy, and her skeleton could not tolerate the increased weight bearing, lumbar lordosis, and physiological changes in bone metabolism that occur during pregnancy.

Osteoporosis in association with pregnancy or lactation is likely under-recognized and under-reported. Approximately 75% of vertebral compression fractures in older women are clinically silent and discovered only through radiological surveys; the same is likely true for reproductive age women. Back pain may signal a vertebral fracture, but it may be readily dismissed as a common symptom of normal pregnancy. Consistent with this, an online survey found that women who suffered compression fractures during pregnancy or lactation experienced a mean delay of 3 months before a diagnostic radiograph was done (87). The medical literature is biased toward reports of women who suffered a frightening cascade of multiple compression fractures in association with pregnancy (79,83), whereas how commonly a single vertebral compression fracture might occur and be detected during pregnancy is unknown. Although the literature has focused on vertebral compression fractures occurring with pregnancy, one report suggested that ankle and other lower limb fractures are more common (88).

Osteoporosis usually presents in association with a first pregnancy (especially during lactation) with many but not all reports suggesting that there is a low risk of recurrence in subsequent pregnancies, and no risk conferred by higher parity (79,80,85,89-92). This may indicate that reversible factors such as nutrition were corrected after the first pregnancy, or that all structurally compromised vertebrae collapsed under the load of the first pregnancy. About 60% of patients present with lower thoracic or lumbar pain that may be quite debilitating due to vertebral collapse (85,91,92). Most cases show normal serum chemistries and calciotropic hormone levels, but in a few, secondary causes of bone loss may be identified, including low calcium intake, anorexia nervosa, celiac disease, hyperparathyroidism, osteogenesis imperfecta, inactivating mutations in LRP5, premature ovarian failure, and corticosteroid or heparin therapy (79,80,86,91-95). For example, a woman’s habitual calcium intake of only 229 mg daily was not enough to meet maternal and fetal demands for calcium, and likely contributed to a cascade of vertebral compression fractures occurring late in pregnancy and the puerperium (79). Bone biopsies have only occasionally been done in women having fractures associated with pregnancy or lactation. Most have confirmed osteoporosis and the absence of osteomalacia, while DXA as shown aBMD Z-scores are often in the low bone mass or osteoporotic ranges (85,91,92).

Pain from vertebral compression fractures resolves spontaneously over several weeks in most cases while the bone density has been reported to substantially improve in most women following pregnancy, including those who fractured. Fractures tend not to recur in subsequent pregnancies. Thus, although myriad medical treatments (bisphosphonates, estrogen, testosterone, calcitonin, teriparatide, denosumab) and surgical interventions (kyphoplasty, vertebroplasty, spinal fusion) have been used in individual cases of pregnancy-associated osteoporosis (79), the tendency for this condition to spontaneously improve may make pharmacological treatment unjustified except for the severest cases. At the least, it may be prudent to wait 12-18 months to determine the extent to which the aBMD recovers on its own after a pregnancy-associated vertebral fracture (79,86).

A recent study examined women with self-reported PLO that resulted in fractures occurring in pregnancy or, more commonly, during lactation (96). They were compared cross-sectionally to two groups of historical controls, healthy premenopausal women and women with known idiopathic osteoporosis (IOP) (96). Women with PLO had lower aBMD and reduced HR-pQCT parameters of cortical and trabecular bone than in healthy women and women with IOP (96). However, women with PLO who were assessed >12 months postpartum (“distant”) had higher aBMD than women who were assessed <12 months postpartum (“early”), which implies that recovery had occurred in the “distant” group (96). Moreover, the aBMD of women in the “distant” group was no different than that of women with IOP (96), which suggests that (at least in this cohort) many women with PLO have low bone mass and strength that precedes pregnancy.

A distinct condition is focal, transient osteoporosis of the hip (79). This is rare, self-limited, and probably not a manifestation of altered calciotropic hormone levels or mineral balance during pregnancy. Instead, it may be a consequence of local factors. A variety of theories have been offered to explain this condition, including femoral venous stasis due to pressure from the pregnant uterus, Sudeck’s atrophy or reflex sympathetic dystrophy (causalgia), ischemia, trauma, viral infections, marrow hypertrophy, immobilization, and fetal pressure on the obturator nerve. These patients present with unilateral or bilateral hip pain, limp and/or hip fracture in the third trimester or puerperium (79,97-99). Radiographs and DXA indicate radiolucency and reduced bone density of the symptomatic femoral head and neck, while MRI demonstrates increased water content of the femoral head and the marrow cavity; a joint effusion may also be present. The differential diagnosis of this condition includes inflammatory joint disorders, avascular necrosis of the hip, bone marrow edema, and reflex sympathetic dystrophy. It is a self-limiting condition with both symptoms and radiological appearance resolving within two to six months post-partum; conservative measures including bed rest are usually all that is required during the symptomatic phase (79). Of course, fractures of an involved femur require urgent arthroplasty or hip replacement. The condition recurs in about 40% of cases (not necessarily during pregnancy), unlike osteoporosis involving the spine, and this has prompted prophylactic hip arthroplasty to be done in a few cases where the opposite hip appears to be affected.

Vertebral compression fractures and transient osteoporosis of the hip are not always distinct entities; both have occurred in a few women in association with pregnancy (100-103).

TREATMENT CONSIDERATIONS

For fragility fractures occurring in association with pregnancy, treatment should include optimization of calcium and vitamin D intake, encouraging judicious weight-bearing physical activity, correction of nutritional deficiencies, and treatment of any reversible causes of bone loss or fragility. A supportive corset may provide short-term pain relief. Breastfeeding is not contraindicated but its relative safety should be discussed, since it will lead to progressive loss in aBMD and a transient further increase in fracture risk (see lactation section, below). The potential to rush in with pharmacotherapy should be tempered by the realization that aBMD normally increases 20-70% during the subsequent six to twelve months in women who fractured but received no interventions (1,79,80,85,86,104-112). Therefore, it seems prudent to delay any use of pharmacotherapy for 12-18 months until the extent of spontaneous recovery has been assessed. The extent of spontaneous recovery of lumbar spine aBMD at 12–18 months should be assessed by DXA. HR-pQCT will underestimate the extent of recovery unless the parameters are adjusted to detect and to capture the newly formed bone (osteoid and under-mineralized bone) (86).

Documented pharmacotherapies for PLO have included calcitonin, bisphosphonates, denosumab, strontium ranelate, and teriparatide, using the same regimens as for post-menopausal osteoporosis but with treatment durations from 6 months to as much as 10 years (1,79,83,93,113-117). These reports are observational and lacked controls to determine whether any improvements in aBMD exceeded what would have been observed with spontaneous recovery (i.e., use of calcium and vitamin D replacement only). A recent systematic review found that teriparatide increased aBMD by 8-37% and bisphosphonates by 3 to 43% (118). Notably, these ranges overlap with the bone density increases of 20-70% achieved by women in other reports who did not receive pharmacotherapy. In the few case series where pharmacologically treated women were compared to women who received calcium and vitamin D supplementation only, the final aBMD did not differ between groups (83,109,119,120). Of greater concern is that in the largest of these studies (107 women), recurrent fractures occurred twice as often in women who received teriparatide or alendronate (or both), as compared to those who received only calcium and vitamin D supplementation (109). That finding suggests that pharmacotherapy might be harmful, but better controlled studies are needed to be certain of the benefits vs. risks of pharmacotherapy in this setting.

Vertebroplasty and kyphoplasty have also been used to treat painful vertebral fractures post-partum, but their overall efficacy is uncertain, given that blinded randomized trials have found no superiority over sham surgery or medical approaches in older subjects (121).

For transient osteoporosis of the hip that has not yet resulted in a fracture, the main consideration is whether to prophylactically rod the affected femur(s), or to observe the patient with the expectation that full spontaneous recovery will occur.

Primary Hyperparathyroidism

This is an uncommon condition but there are no firm data available on its prevalence. Hypercalcemia has been found in 0.03% of routinely screened reproductive age women, while two case series indicated that 1% of all parathyroidectomies were done during pregnancy (122,123). There are at least several hundred cases in the medical literature. The diagnosis will be obscured by the normal pregnancy-induced changes that lower the total serum calcium and suppress PTH; however, finding the ionized or albumin-corrected calcium to be increased, and PTH to be detectable, should indicate primary hyperparathyroidism in most cases (note the exception of FHH in the next section).

Physiological changes of pregnancy described earlier increase intestinal calcium absorption and bone resorption, and cause hypercalciuria. In turn these developments can worsen primary hyperparathyroidism and may lead to more severe hypercalcemia, pancreatitis, and kidney stones. The potential for worsening of hypercalcemia is also offset in part by active transfer of calcium across the placenta into the developing fetus.

Primary hyperparathyroidism during pregnancy has been reported to cause a variety of symptoms that are not specific to hypercalcemia and cannot be distinguished from those occurring in normal pregnancy (nausea, vomiting, renal colic, malaise, muscle aches and pains, etc.). Conversely the literature has associated primary hyperparathyroidism with an alarming rate of adverse outcomes in the fetus and neonate, including a 10-30% rate for each of spontaneous abortion, stillbirth, and perinatal death, and 30-50% incidence of neonatal tetany (123-127). These high rates were reported in older literature; more recent case series suggest that the rates of stillbirth and neonatal death are each about 2%, while neonatal tetany occurred in 15% (124). The adverse postnatal outcomes are thought to result from suppression of the fetal and neonatal parathyroid glands; this suppression may be prolonged after birth for 3-5 months (124) and in some cases it has been permanent (124,126,128).

To prevent these adverse outcomes, surgical correction of primary hyperparathyroidism during the second trimester has been almost universally recommended. Several case series have found elective surgery to be well tolerated, and to dramatically reduce the rate of adverse events when compared to the earlier cases reported in the literature. In a series of 109 mothers with hyperparathyroidism during pregnancy who were treated medically (N=70) or surgically (N=39), there was a 53% incidence of neonatal complications and 16% incidence of neonatal deaths among medically treated mothers, as opposed to a 12.5% neonatal complications and 2.5% neonatal deaths in mothers who underwent parathyroidectomy (123). A systematic review of 382 cases found that neonatal deaths and infant morbidity were lower in surgically treated vs. medically treated mothers (9.1 vs. 38.9%) (129). Furthermore, among surgically treated mothers, neonatal death and infant morbidity significant reduced with surgery done in the second versus third trimesters (4.5 vs. 21.1%) (129). Choosing the second trimester allows organogenesis to be complete in the fetus and to avoid the poorer surgical outcomes and risk of preterm birth associated with surgery during the third trimester (124,127,130,131).

Many women in the earliest published cases had a more severe form of primary hyperparathyroidism that is not often seen today (symptomatic, with nephrocalcinosis and renal insufficiency). While mild, asymptomatic primary hyperparathyroidism during pregnancy has been followed conservatively with successful outcomes, complications continue to occur, so that, in the absence of definitive data, surgery during the second trimester remains the most common recommendation (132). An analysis of 1,057 reproductive-aged women with primary hyperparathyroidism found that the rate of C-sections was doubled but there was no difference in the incidence of spontaneous abortions; no data were available on other pregnancy outcomes or neonatal complications (133). In another study of 134 pregnancies in women with primary hyperparathyroidism compared to 431 pregnancies in normocalcemic women, there were no differences in pregnancy-related complications or spontaneous abortions, but neonatal complications were not reported (134). Other recent cases are consistent with lower rates of still birth, neonatal death, and neonatal tetany as compared to the older literature. Therefore, it is reasonable that milder cases diagnosed during the third trimester may be observed until delivery; however, rapid and severe postpartum worsening of the hypercalcemia can occur (131,135-138). This postpartum “parathyroid crisis” occurs because the placental calcium outflow has been lost, while surging PTHrP production in the breasts means an additional factor stimulating bone resorption.

TREATMENT CONSIDERATIONS

The main consideration is whether to operate electively in the second trimester or observe the patient in the hope that surgical intervention can be delayed until after delivery.

Of the five international consensus conferences on the management of primary hyperparathyroidism, only the most recent one commented on pregnancy (139). There are no definitive medical management guidelines for hyperparathyroidism during pregnancy apart from ensuring adequate hydration and correction of electrolyte abnormalities (132). There is some consensus that surgery is indicated for a persistent serum calcium above 2.80 mmol/L (11.1 mg/dL), or an ionized calcium above 1.4 mmol/L (5.6 mg/dL) (140,141). However, another review suggested a higher level 3.00 mmol/L (12.0 mg/dL) (142). If surgery is undertaken, a bilateral approach is often warranted because of the lack of preoperative imaging to localize the adenoma.

Pharmacologic agents to treat hypercalcemia have not been adequately studied in pregnancy, and follow-up on the babies has been brief (if at all). Calcitonin does not cross the placenta and has been used safely (132). Oral phosphate has also been used but is limited by diarrhea, hypokalemia, and risk of soft tissue calcifications. Bisphosphonates are relatively contraindicated because of their potential adverse effects on fetal endochondral bone development, although a review of 78 cases of bisphosphonate use in pregnancy found no obvious problems in most cases (112). Denosumab crosses the placenta and has been shown to cause an osteopetrotic-like phenotype in fetal cynomolgus monkeys and rats (143,144), and so it should be avoided in human pregnancy. High-dose magnesium has been proposed as a therapeutic alternative which should decreases serum PTH and calcium levels by activating the calcium sensing-receptor, but it has not been adequately studied for this purpose (145,146). The calcium receptor agonist cinacalcet, which is used to suppress PTH and calcium in nonpregnant subjects with primary or secondary hyperparathyroidism and parathyroid carcinoma, has also been tried in pregnancy (147-150). However, since the calcium receptor is expressed in the placenta and regulates fetal-placental calcium transfer (151), the possibility of adverse effects of cinacalcet on the fetus and neonate remain a concern. In 6 case reports, use of calcimimetics resulted in neonatal hypocalcemia in half of them (142). Heparin-free hemodialysis can lower the serum calcium before surgery (152). The recent consensus conference on management of primary hyperparathyroidism advised against the use of bisphosphonates and denosumab and cautioned that data on use of cinacalcet are very limited (139). Nevertheless, these agents have been used when there is a hypercalcemic crisis and surgery isn’t possible; the author is aware of cases treated pharmacologically that have not been reported in the literature.

In any case that was followed medically, parathyroidectomy is recommended to be done postpartum, with monitoring in place to detect a postpartum hypercalcemic crisis. Since these women are presenting young with primary hyperparathyroidism, genetic testing may be indicated to rule out inherited causes.

Familial Hypocalciuric Hypercalcemia (FHH)

Inactivating mutations in the calcium-sensing receptor cause this autosomal dominant condition which presents with hypercalcemia and hypocalciuria (153). During pregnancy there will be persistent hypercalcemia with non-suppressed PTH, and the serum calcium may progressively rise across the trimesters. As noted above, fractional excretion of calcium is not reduced during pregnancy in this condition, because it is overridden by the physiological increase in intestinal calcium absorption that in turn causes hypercalciuria (50,51,154). Consequently, FHH presenting during pregnancy can be easily mistaken for primary hyperparathyroidism. Unfortunately, at least one pregnant woman with FHH was mistaken to have primary hyperparathyroidism because of worsening hypercalcemia and hypercalciuria and underwent a three-and-a-half gland parathyroidectomy during the second trimester. FHH was only recognized when her hypercalcemia persisted and her neonate was found to be hypercalcemic too (50).

Pregnancy in women with familial hypocalciuric hypocalcemia should be uneventful for the mother, but the maternal hypercalcemia has caused fetal and neonatal parathyroid suppression with subsequent tetany in both normal and hemizygous children (5,155,156). A hemizygous neonate will later develop benign hypercalcemia, but if the baby has two inactivating mutations of the calcium receptor (most commonly from both parents being hemizygous for FHH), then the neonate may suffer a life-threatening hypercalcemic crisis (5).

TREATMENT CONSIDERATIONS

Hypercalcemia is a normal state of affairs for women with FHH and it should not be treated or mistaken for primary hyperparathyroidism. Instead, the newborn should be watched for postnatal hypocalcemia and for the later development of hypercalcemia as a sign that it inherited the mutation.

Hypoparathyroidism

Hypoparathyroidism during pregnancy usually presents as a pre-existing condition that the clinician is challenged to manage. The natural history of hypoparathyroidism during pregnancy is confusing due to seemingly conflicting case reports in the literature [reviewed in (1,3,157,158)]. Early in pregnancy, some hypoparathyroid women have fewer hypocalcemic symptoms and require less supplemental calcium. This is consistent with a limited role for PTH in the pregnant woman and suggests that an increase in calcitriol and/or increased intestinal calcium absorption occurs in the absence of PTH. However, other case reports clearly indicate that some pregnant hypoparathyroid women required increased calcitriol replacement in order to avoid worsening hypocalcemia. Adding to the confusion is that in some case reports, it appears that the normal, artifactual decrease in total serum calcium during pregnancy was the parameter that led to treatment with increased calcium and calcitriol supplementation; fewer cases reported that dose increments in calcitriol and calcium were made because of maternal symptoms of hypocalcemia or tetany, or objective evidence of true hypocalcemia (low ionized or albumin-corrected calcium).

In a well-documented case, calcitriol was stopped and the woman required only supplemental calcium during the third trimester; she developed hypocalcemia within 48 hours of delivery, which implicates loss of placental PTHrP as contributing to her normalization during pregnancy (159). In a series of ten cases of hypoparathyroidism, the ionized calcium remained normal during pregnancy with no need for calcitriol (160).

Among these and other recent cases, it is clear that hypoparathyroidism may improve, stay the same, or even worsen during pregnancy (159,161-163). It is not possible to know in advance who will improve and who will worsen during pregnancy; the task is to maintain the albumin-corrected serum calcium or ionized calcium in the normal range for the duration of pregnancy. Maternal hypocalcemia due to hypoparathyroidism must be avoided because it has been associated with intrauterine fetal hyperparathyroidism and fetal death. Conversely, overtreatment must be avoided because maternal hypercalcemia is associated with the fetal and neonatal complications described above under Primary Hyperparathyroidism. Calcitriol and 1α-calcidiol are recommended due to their shorter half-lives, lower risk of toxicity, and the clinical experience with these agents.

Late in pregnancy, hypercalcemia may occur in hypoparathyroid women unless the calcitriol dosage and supplemental calcium are substantially reduced or discontinued. This effect appears to be mediated by the increasing levels of PTHrP in the maternal circulation in late pregnancy. Conversely, one case report of hypoparathyroidism in pregnancy found that there was a transient interval of increased requirement for calcitriol immediately after delivery and before lactation was fully underway (159). This may be the result of loss of placental sources of PTHrP followed by a surge in production of PTHrP by the lactating breast (see lactation section, below).

TREATMENT CONSIDERATIONS

The albumin-corrected serum calcium should be maintained in the mid-normal range in order to insure adequate delivery of calcium to the fetus. This short-term recommendation differs from the common recommendation for non-pregnant adults of maintaining the albumin-corrected serum calcium near or just below the lower end of normal, which reduces the renal filtered load and may slow the progression of nephrocalcinosis over the long term. As discussed above, management during pregnancy may not require any change in pre-existing doses of calcium and calcitriol or 1α-calcidiol, or it may require increases or decreases in both the calcium and the active vitamin D analog.

Pseudohypoparathyroidism

Pseudohypoparathyroidism is a genetic disorder causing resistance to PTH and manifest by hypocalcemia, hypophosphatemia, and high PTH levels. The two main subtypes include type I, which has blunted PTH-induced phosphaturia and renal production of cyclic AMP, while type II has blunting of PTH-induced phosphaturia only. They are managed similarly to hypoparathyroidism.

The published experience with pseudohypoparathyroidism during pregnancy is similar to that of hypoparathyroidism, with a mix of cases that improved, worsened, or had no change. Type I pseudohypoparathyroidism improved during four pregnancies as shown by fewer hypocalcemic symptoms, achievement of normocalcemia, lowering of PTH to near-normal, calcitriol increasing several-fold, urinary calcium excretion normalizing, and supplemental vitamin D, calcitriol, or analogs no longer required (164). These findings are consistent with PTH-independent increases in intestinal calcium absorption and calcitriol synthesis occurring during pregnancy that in turn improve calcium homeostasis; endogenous serum calcitriol did double mid-pregnancy in two women in whom supplemental calcitriol had been discontinued. However, in seven other pregnancies in women with types I and II pseudohypoparathyroidism there was subjective worsening of hypocalcemia-like symptoms, or the apparent need to increase the doses of calcium, calcitriol, or 1α-calcidiol (1,165-168). Another case reported that no change in calcium or calcitriol dosages were required during pregnancy (169). Lastly, a more recent series of 5 patients reported variability of improved, worsened, or no change in the condition during pregnancy (170).

If maternal hypocalcemia persists during pregnancy, pseudohypoparathyroidism can lead to the same adverse fetal outcomes that have been associated with maternal hypoparathyroidism, including parathyroid hyperplasia, skeletal demineralization, and fractures (171,172). The maternal calcium concentration must be maintained in the normal range to avoid these fetal outcomes.

TREATMENT CONSIDERATIONS

Maintain the albumin-corrected serum calcium in the mid-normal range. As with hypoparathyroidism, this may not require any change in pre-existing doses of calcium and calcitriol or 1α-calcidiol, or it may require increases or decreases in both the calcium and active vitamin D analog.

Pseudohyperparathyroidism

As mentioned above, pseudohyperparathyroidism is hypercalcemia that is caused by physiological release of PTHrP driving increased skeletal resorption, akin to how PTHrP also causes hypercalcemia of malignancy. In several cases the breasts were the confirmed source of PTHrP because the hypercalcemia and elevated PTHrP did not abate until a bilateral reduction mammoplasty was carried out (173-175). The condition has occurred in women who simply have large breasts (175-177). In another case the hypercalcemia, elevated PTHrP, and suppressed PTH reversed within a few hours of an urgent C-section, thereby confirming the placenta as the source (178). In all cases of pseudohyperparathyroidism, it should be anticipated that the cord blood calcium will also be increased, and that the baby is at risk for fetal and neonatal hypoparathyroidism with hypocalcemic tetany.

TREATMENT CONSIDERATIONS

The diagnosis may not be clear until after delivery, when the serum calcium rapidly normalizes (indicating placental PTHrP was the cause) or stays elevated (indicating production of PTHrP by the breasts is the cause). Prior to delivery, medical management is similar to that for primary hyperparathyroidism.

Vitamin D Deficiency and Insufficiency

There are no comprehensive studies of the effects of vitamin D deficiency or insufficiency on human pregnancy, but the available data from small clinical trials of vitamin D supplementation, observational studies, and case reports suggest that, consistent with animal studies, vitamin D insufficiency and deficiency is not associated with any worsening of maternal calcium homeostasis (this topic is reviewed in detail in (1,4,7). Maternal hypocalcemia is milder with vitamin D deficiency due to the effects of secondary hyperparathyroidism to increase skeletal resorption and renal calcium reabsorption. Consequently, hypocalcemia due to vitamin D deficiency has not been clearly associated with the same adverse fetal outcomes that maternal hypoparathyroidism causes (reviewed in detail in (5,179)). The fetal effects of vitamin D deficiency, inability to form calcitriol, and absence of the vitamin D receptor have been examined across several animal species and all have indicated that the fetus will have a normal serum calcium and fully mineralized skeleton at term (reviewed in detail in (5,179)). Neonatal hypocalcemia and rickets can occur in infants born of mothers with severe vitamin D deficiency, but it is usually in the weeks to months after birth that this presents, after intestinal calcium absorption becomes dependent on calcitriol.

There has been much interest in studies that have inconsistently associated third-trimester measurements of 25OHD, or estimated vitamin D intakes during pregnancy or the first year after birth, with possible extraskeletal benefits in the mother (reduced bacterial vaginosis, pre-eclampsia, pre-term delivery) or in the offspring (lower incidence of type 1 diabetes, greater skeletal mineralization, etc.). These associational studies won’t be discussed in detail (some are cited in: (1,5,180)) because they are confounded by factors which contribute to lower 25OHD levels (maternal overweight/obesity, lower socioeconomic status, poor nutrition, lack of exercise, etc.). It is necessary to test these associations in randomized clinical trials that compare higher versus lower intakes of vitamin D during pregnancy. At present the results of the associational studies are insufficient to warrant prescribing higher intakes of vitamin D during pregnancy to prevent these postulated outcomes.

Among many clinical trials of vitamin D supplementation that have been carried out (1), only a few have included over a 100 study participants who were vitamin D deficient at entry, while other recent studies that gained press attention did not include many vitamin D deficient subjects at all.

Among the trials with over 100 participants (14,181-189), the two largest were from Bangladesh and UK with over 1,000 participants (187,188). Baseline maternal 25OHD levels were lowest (20-29 nmol/L) in trials from Bangladesh, UK, Iran, and UAE, and in the 40-60 nmol/L range in the remainder. Interventions consisted of placebo/no treatment versus low dose (400 IU/day) or high dose (1,000-5,000 IU/day) vitamin D supplementation, initiated before mid-pregnancy, and maintained until delivery. For most trials the primary outcomes were simply maternal and neonatal-cord blood 25OHD and calcium. The most recent and largest study was from Bangladesh, and the primary outcome was pre-specified as infant length-for-age z-scores at 1 year of age (188). Offspring anthropometric parameters and/or bone mineral content were pre-specified only in a few of the remaining studies (184,186,187).

In all studies vitamin D supplementation increased maternal serum and cord blood 25OHD, but there was no overall effect on maternal or cord blood calcium, except one trial that showed a small but significant difference in maternal calcium at delivery (181). The largest achieved difference in maternal 25OHD was over 160 nmol/L (60 ng/mL) in a single study: 16 nmol/L (6.4 ng/mL) in untreated and 168 nmol/L (67 ng/mL) in vitamin D-supplemented mothers at term; however, there was no obstetrical or fetal benefit (181). In that study, the incidence of neonatal hypocalcemia was reduced in offspring of vitamin D treated mothers, reflecting the role of calcitriol to stimulate postnatal intestinal calcium absorption (181). Three trials (mean baseline 25OHD of 20-56 nmol/L) showed a significantly higher maternal PTH level in the low dose vitamin D/placebo arms compared to the high dose arms (182-184), while another trial (mean baseline 25OHD 58-60 nmol/L) did not show any effect of vitamin D supplementation on maternal PTH (14). Vitamin D supplementation also had no effect on maternal aBMD parameters in the post-partum period (14,186,190). Four trials assessed obstetrical outcomes (gestational hypertension, pre-eclampsia, gestational diabetes, infection, post-partum hemorrhage, preterm labor and others), and found no effect of vitamin D supplementation (183,187-189). However, one trial from Iran that showed a 54% risk reduction in gestational diabetes when vitamin D was taken at a dose of 50,000 IU every 2 weeks and compared to 400 IU per day (189).

In the large Bangladesh study, there were no significant differences in infant anthropometrics or any other fetal, neonatal or maternal outcomes (188). In one US-based study there was no benefit on mode of delivery, gestational age at delivery, and preterm birth (14), while in another there was no benefit on mode of delivery, C-section rates, adverse events, hypertension, infection, gestational diabetes, still birth, gestational age at delivery, or combinations of these outcomes (183). The UK MAVIDOS trial reported no obstetrical benefit, and no benefit to any of the primary (neonatal bone area, BMC, and aBMD within the first 10-14 days after birth) or secondary outcomes (anthropometric and body composition parameters within 48 hours of birth). However, it received much publicity for a demonstrated increase in BMC and aBMD in winter-born neonates of vitamin D-supplemented vs. placebo-treated mothers (187). Because the neonatal skeleton accretes 100 mg/day of mineral content after birth, this result may reflect improved intestinal mineral delivery over 14 days after birth, rather than a prenatal effect on skeletal mineralization (1,191,192). Curiously, autumn-born neonates of vitamin D-supplemented vs. placebo-treated mothers showed an adverse trend of similar magnitude on BMC and aBMD, which suggests possible harm from vitamin D supplementation, or chance findings due to small numbers within the sub-groups (192). These sub-group analyses of treatment by season interaction were not specified outcomes in the trial registries (ISRCTN 82927713 and EUDRACT 2007-001716-23). In the UK study that achieved the greatest difference in 25OHD levels between untreated and vitamin D-treated mothers and babies, there was a trend for more small for gestational age babies born to mothers who did not receive antenatal vitamin D supplementation (28% vs. 15%, p<0.1), but the study was not powered for this outcome (181). In studies from the UAE, and Iran there was also no benefit on obstetrical outcomes (variably, mode of delivery, C-section rates, adverse events, stillbirths, gestational age at delivery) or neonatal anthropometric measurements and bone mass measurements (182,184,186).

The lack of any beneficial effect on maternal, immediate fetal/neonatal and neonatal outcomes (anthropometrics and cord blood calcium), even in studies that included mothers with some of the lowest 25OHD levels (181,184,186,188), suggests that vitamin D supplementation during pregnancy confers no benefit to the neonate. The most recent study was well-powered to demonstrate a beneficial effect on infant length and other fetal/neonatal outcomes, but did not yield any significant results, despite low vitamin D levels in the mothers at study entry (188).

Multiple systematic reviews have assessed the effect of vitamin D supplementation during pregnancy on maternal outcomes (193-198). The reviews differed significantly in their methodology, eligibility criteria, intervention (vitamin D alone or combined with calcium), and inclusion of trials. The Cochrane systematic review showed no significant effect of vitamin D supplementation alone on obstetrical outcomes, but there was a significant reduction in preterm birth and low birth weight (194). Conversely, the combination of vitamin D with calcium was found to reduce the risk of pre-eclampsia, but at the cost of a potential increase in preterm births (194). The other reviews yield variable and inconsistent results among them (193,195-198).

An update to the Cochrane systematic review compared vitamin D regimens during pregnancy (≤600 IU versus >600 IU per day; <4000 IU versus ≥ 4000 IU per day) (199). There was no significant effect on pre-eclampsia, preterm birth and low birth weight (low/very low quality of the evidence) (199). The risk of gestational diabetes was reduced with vitamin D supplementation in only one comparison (moderate quality evidence), favoring a dose >600 IU/d (RR 0.54 (0.34 to 0.86). The reviewers noted a concern that safety data were often not reported in the trials that included high doses of vitamin D (199).

In summary, the few large RCTs reported to date do not provide evidence for a beneficial effect of high dose vitamin D supplementation (1,000-5,000 IU/day), on maternal and neonatal outcomes. A recent large trial makes it unlikely that vitamin D supplementation in deficient women would yield any beneficial effect on infant length (188). Other studies had low power, baseline maternal serum 25OHD levels that were often not low, and lack of pre-specification of obstetrical and neonatal outcomes. A potential protective effect of vitamin D on neonatal BMC observed in the MAVIDOS trial (187)makes physiological sense because the intestines become the route of calcium delivery after birth, and the finding is consistent with the benefit observed in some RCTs of prenatal vitamin D supplementation reducing the incidence of neonatal hypocalcemia.

Overall, available data are insufficient from the individual clinical trials or these systematic reviews to conclude that vitamin D supplementation during pregnancy confers any proven obstetrical benefits, especially with respect to calcium and bone metabolism. However, much interest remains in determining whether vitamin D prevents adverse non-skeletal events in mother and baby. In the meantime, one should always ensure that any pregnant woman is vitamin D sufficient prior to pregnancy, or soon after her pregnancy is confirmed. This ensures that the baby is born with adequate 25OHD so that calcitriol can be upregulated postnatally to stimulate calcium and phosphate absorption in the neonatal intestines.

TREATMENT CONSIDERATIONS

Vitamin D supplementation is not harmful to the nonpregnant adult unless excessive doses are administered that cause hypervitaminosis D (typically in excess of 10,000 IU daily); however, the maximal level of maternal intake that is safe for the developing fetus has not been established. Clinical trials in pregnant women have safely administered doses ranging from 400 to 5,000 IU of vitamin D daily without obvious adverse effects to mother or offspring. All pregnant women should have their vitamin D intake optimized. This should prevent any non-skeletal outcomes that may be caused by vitamin D deficiency and will also ensure that the newborn has sufficient vitamin D stores to be able to normalize mineral homeostasis in the hours to days after birth, when it switches from being dependent upon the placenta for mineral delivery to relying on its maturing intestines to absorb that mineral from milk.

Genetic Vitamin D Resistance Syndromes

Case reports and series have provided insight into the effect of pregnancy on genetic disorders of vitamin physiology. Pregnancies have generally been unremarkable in women with vitamin D-dependent rickets type 1 (VDDR-I) which is due to absence of Cyp27b1, and in women with VDDR-II that is due to absence of functional VDRs (200-202). In one such uneventful VDR-II pregnancy, the pre-pregnancy intake of supplemental calcium (800 mg) and high-dose calcitriol were maintained until her clinicians increased the dose of calcitriol later in pregnancy “because of the knowledge that the circulating 1,25-(OH)₂D concentration normally rises during pregnancy,” and not because of any change in albumin-adjusted serum calcium (201). Consequently, it’s unclear whether any change was needed. However, it is reasonable to increase the dose of calcitriol to mirror the increase that happens during normal pregnancy. In women with VDDR-I, the dose of calcitriol was unchanged in one-third of pregnancies but increased 1.5 to 2-fold in others (200).

TREATMENT CONSIDERATIONS

Maintain a normal albumin-corrected serum calcium with adjustments to oral calcium and calcitriol dosing as needed based on serial monitoring of blood chemistries.

24-Hydroxylase Deficiency

Genetic loss of the catabolic effects of 24-hydroxylase (CYP24A1) causes high calcitriol, mild hypercalcemia, and nephrolithiasis in non-pregnant adults, which may be asymptomatic (203). But during pregnancy in affected individuals, the physiological 2 to 5-fold increase in calcitriol is unopposed by catabolism, which causes an exaggerated increase in calcitriol, followed by severe and potentially life-threatening hypercalcemia. This has been confirmed by study of a mouse model of Cyp24a1 ablation that mimics the human condition (204). In affected women, hypercalcemia can be quite marked, with suppressed or undetectable PTH, and calcitriol concentrations that exceed what is expected for pregnancy (205-207). Pregnant patients may also present with nephrolithiasis or acute pancreatitis (207,208).

TREATMENT CONSIDERATIONS

Treatment of hypercalcemia is difficult because the agents that could be used are not approved for pregnancy. Increased intestinal calcium absorption is the direct cause, and so use of increased hydration and a modestly restricted calcium diet, combined with phosphate supplementation to bind dietary calcium, are relatively safe management approaches. If PTH increases above normal, then dietary calcium restriction should be lessened to prevent maternal bone resorption and fetal secondary hyperparathyroidism. Other pharmacologic therapy should be reserved for the most severe cases and used with caution. This includes oral glucocorticoids to suppress intestinal calcium absorption, loop diuretics, calcitonin, and bisphosphonates; denosumab should not be used because of teratogenic effects observed in cynomolgus monkeys and mice (143,144). Cinacalcet will not be useful because PTH will already be suppressed due to the combined effects of pregnancy and hypercalcemia.

More targeted treatments include ketoconazole or other azoles to inhibit calcitriol synthesis (209), or rifampin to stimulate catabolism of calcitriol via the 23-hydroxylase pathway (209,210). These drugs have been used in pregnancy to treat other conditions, and so there are data to support their relative safety. However, no case reports have yet involved use of these drugs to treat 24-hydroxylase deficiency in pregnancy.

Low or High Calcium Intake

Through the doubling of intestinal calcium absorption during pregnancy, women have the ability to adapt to wide ranges of calcium intakes and still meet the fetal demand for calcium. It is conceivable that extremely low maternal calcium intakes could impair maternal calcium homeostasis and fetal mineral accretion, but there are scant clinical data examining this possibility (211). One prospective study found that a dietary calcium intake of less than 800 mg daily during the third trimester was associated with significantly lower aBMD at 5 years post-pregnancy (212). Among women with low dietary calcium intake, there are differing results as to whether or not calcium supplementation during pregnancy improved maternal or neonatal bone density (213). There is short term evidence that bone turnover markers were reduced when 1.2 gm of supplemental calcium was given for 20 days to 31 Mexican woman at 25-30 weeks of gestation; their mean dietary calcium intake was 1 gm (214). In a double-blind study conducted in 256 pregnant women, 2 gm of calcium supplementation improved bone mineral content only in the infants of supplemented mothers who were in the lowest quintile of calcium intake (215). Among cases of fragility fractures presenting during pregnancy, some women had very low calcium intakes (<300 mg per day), and in such cases substantial maternal skeletal resorption must be invoked in order to meet the fetal calcium requirement and maintain the maternal serum calcium concentration (79).

Overall the physiological changes in calcium and bone metabolism that usually occur during pregnancy and lactation are likely to be sufficient for fetal bone growth and breast-milk production in women with reasonably sufficient calcium intake (216). However, the use of calcium supplementation for pregnant women with low calcium intake can be defended by the links between low calcium intake and both preeclampsia and hypertension in the offspring (211). Clinical trials and meta-analyses have also demonstrated the supplemental calcium will reduce the risk of preeclampsia in women with low dietary calcium intakes, but not in those with adequate intake (217-220).

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

TREATMENT CONSIDERATIONS

Very low calcium intake must be avoided because it increases the risk of pre-eclampsia, maternal skeletal resorption, and inadequate mineralization of the fetal skeleton. Conversely, high calcium intake must be avoided because it increases the risk of maternal hypercalcemia and suppression of the fetal parathyroids. The Institute of Medicine advises that pregnant women require the same calcium intake as non-pregnant women, a value that ranges from 1,000 to 1,200 mg daily, depending on age (221).

Hypercalcemia of Malignancy

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

FGF-23 Disorders

X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene, which lead to high circulating levels of FGF23. In turn this causes hypophosphatemia with rickets or osteomalacia. Pregnancies were normal in a mouse model of XLH. In particular, despite very high circulating levels of FGF23, which normally downregulate calcitriol synthesis and increase its catabolism, maternal serum calcitriol increased to the high levels normally seen during pregnancy (19,222). This rise in calcitriol should contribute to increased intestinal calcium and phosphate absorption. Several case reports documented persistent hypophosphatemia during pregnancy in women with XLH, but no adverse outcomes (223,224). Nevertheless, it is generally recommended to supplement with calcitriol and phosphate to keep the serum phosphate near normal during pregnancy.

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

TREATMENT CONSIDERATIONS

For XLH and other FGF-23 mediated disorders that lead to hypophosphatemia, the serum phosphate should be kept near normal with the use of phosphate supplements and calcitriol if needed. Burosumab is a new anti-FGF23 antibody that corrects hypophosphatemia in XLH and tumor-induced hypophosphatemia (oncogenic osteomalacia). It should not be used in pregnancy because preclinical studies have shown that it crosses the placenta and causes toxicity in cynomolgus monkeys (placental mineralization, late fetal loss, shortened gestation, and preterm births) (225).

For hyperphosphatemic disorders due to inadequate FGF23 action, there are no data to guide potential treatment guidelines. However, judicious use of phosphate binders may be of value, with avoidance of any that may be harmful to the fetus.

MINERAL PHYSIOLOGY DURING LACTATION AND POST-WEANING

As lactation begins the mother is faced with another demand for calcium in order to make milk. The average daily loss of calcium into breast milk is 210 mg, although daily losses as great as 1000 mg calcium have been reported is some women nursing twins (1). Although women meet the calcium demands of pregnancy by upregulating intestinal calcium absorption and serum concentrations of calcitriol, a different adaptation occurs during lactation. A temporary resorption and demineralization of the maternal skeleton appears to be the main mechanism by which breastfeeding women meet these calcium requirements. This adaptation does not appear to require PTH or calcitriol but is regulated by the combined effects of increased circulating concentrations of PTHrP and low estradiol levels. Characteristic changes in serum minerals are calciotropic hormones are depicted in Figure 3.

Figure 3. Schematic depiction of longitudinal changes in calcium, phosphorus, and calciotropic hormone levels during lactation and post-weaning skeletal recovery in women. Normal adult values are indicated by the shaded areas. PTH does not decline in women with low calcium or high phytate intakes and may even rise above normal. Calcidiol (25OHD) values are not depicted; most longitudinal studies indicate that the levels are unchanged by lactation but may vary due to seasonal variation in sunlight exposure and changes in vitamin D intake. PTHrP and prolactin surge with each suckling episode, and this is represented by upward spikes. FGF23 values cannot be plotted due to lack of data. Very limited data suggest that calcitriol and PTH may increase during post-weaning, and the lines are dashed to reflect the uncertainty. Reproduced with permission from (1).

Mineral Ions

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

Parathyroid Hormone

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

Vitamin D Metabolites

A common concern has been that the suckling neonate will deplete maternal 25OHD stores, but this is not the case. 25OHD should not decline because it does not enter breast milk; conversely, although vitamin D can enter milk, it is present at very low concentrations because appreciable amounts exist in the maternal circulation for only a short postprandial interval. In observational studies and in the placebo arms of several clinical trials, there was either no change or at most a nonsignificant decline in maternal 25OHD levels during lactation, even in severely vitamin D deficient women (4). Calcitriol levels were twice normal during pregnancy but both free and bound calcitriol levels fall to normal within days of parturition and remain there in breastfeeding women (a single study found that women breastfeeding twins had higher calcitriol concentrations than women nursing singletons) (226). Animal studies show that severely vitamin D deficient rodents and mice lacking the vitamin D receptor are able to lactate and provide normal milk (4,47), thereby indicating that vitamin D and calcitriol are not required for lactation to proceed normally (at least in rodents). However, a more recent study found that mice lacking calcitriol produced milk with a lower calcium content (23).

Calcitonin

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

PTHrP

Plasma PTHrP concentrations are significantly higher in lactating women than in non-pregnant controls. The source of PTHrP appears to be the breast, which secretes PTHrP into breast milk at concentrations that are 1,000 to 10,000 times the level found in the blood of patients with hypercalcemia of malignancy or in normal human controls. The circulating PTHrP concentration also increases after suckling (227,228). Additional evidence that the breasts are the source of PTHrP include that ablation of the PTHrP gene selectively from mammary tissue resulted in reduced circulating levels of PTHrP in lactating mice (229). PTHrP also has an intimate association with breast tissue: in animals it has been shown to regulate mammary development and blood flow, and the calcium and water content of milk in rodents, whereas in humans it is commonly expressed by breast cancers.

Furthermore, as described in more detail below, during lactation PTHrP reaches the maternal circulation from the lactating breast to cause resorption of calcium from the maternal skeleton, renal tubular reabsorption of calcium, and (indirectly) suppression of PTH. In support of this hypothesis, deletion of the PTHrP gene from mammary tissue at the onset of lactation resulted in more modest losses of bone mineral content during lactation in mice (229). In humans, PTHrP correlates with the amount of bone mineral density lost, negatively with serum PTH, and positively with the ionized calcium of lactating women (227,230,231). Lastly, clinical observations in hypoparathyroid and aparathyroid women demonstrate the physiological importance of PTHrP to regulate calcium and skeletal homeostasis during lactation (see Hypoparathyroidism, below).

Prolactin

Prolactin is persistently elevated during early lactation and spikes further upward with suckling. Later during lactation basal prolactin levels are normal but continue to spike with suckling. Prolactin is important for initiating and maintaining milk production (232), but it also alters bone metabolism by stimulating PTHrP production in lactating mammary tissue, inhibiting GnRH and ovarian function, and possibly (as noted earlier) through direct actions in osteoblasts that express the prolactin receptor.

Oxytocin

Oxytocin induces milk ejection by contracting myoepithelial cells within mammary tissue. If milk is not ejected, the pressure of milk stasis causes apoptosis of mammary cells, and lactation ceases. Oxytocin spikes in the maternal circulation within 10 minutes after the start of suckling (233). As noted earlier, the oxytocin receptor is expressed in osteoblasts and osteoclasts. But whether oxytocin plays a role in bone metabolism during lactation has proven difficult to determine because oxytocin null mice cannot lactate due to the lack of milk ejection (234).

Estradiol

In lactating women, estradiol levels fall to menopausal levels or below. This stimulates RANKL and inhibits osteoprotegerin production by osteoblasts, thereby stimulating osteoclast proliferation, function, and bone resorption. Studies in mice have shown that increasing the serum estradiol concentration to 7 times the virgin level blunts the magnitude of bone loss during lactation (235), which confirms that estradiol deficiency plays a role in the skeletal resorption that occurs during lactation.

FGF23

A single longitudinal study found that intact FGF23 approximately doubled between late pregnancy and weeks 14 and 26 of lactation (34). An increase in FGF23 may occur in response to the increased bone resorption during lactation, which leads to a higher serum phosphorus (1).

Other Hormones

Serotonin appears to be involved in regulating PTHrP and its effect to resorb the maternal skeleton (236,237). Lactation induces changes in myriad other hormones, such as luteinizing and follicle stimulating hormone, progesterone, testosterone, inhibins, and activins. Whether these play roles in regulating skeletal metabolism during lactation has not been investigated.

Intestinal Absorption of Calcium and Phosphate

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

Intestinal phosphate absorption has not been measured during human lactation, whereas in rodents it remains increased (1).

Renal Handling of Calcium and Phosphate

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

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

Skeletal Calcium Metabolism and Bone Density/Bone Marker Changes

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

Serial measurements of aBMD during lactation (by SPA, DPA or DXA) have shown that bone mineral content falls 3 to 10.0% in women after two to six months of lactation at trabecular sites (lumbar spine, hip, femur and distal radius), with smaller losses at cortical sites and whole body (1,60). These aBMD changes are in accord with studies in rats, mice, and primates in which the skeletal resorption has been shown to occur largely at trabecular surfaces and to a lesser degree in cortical bone, and as much as 25-30% of bone mass or aBMD is lost during three weeks of lactation in normal rodents. The loss in women occurs at a peak rate of 1-3% per month, far exceeding the 1-3% per year that can occur in postmenopausal women who are considered to be losing bone rapidly. This bone resorption is an obligate consequence of lactation and cannot be prevented by increasing the calcium intake in women. Several randomized trials and other studies have shown that calcium supplementation does not significantly reduce the amount of bone lost during lactation (238-241). Not surprisingly, the lactational decrease in bone mineral density correlates with the amount of calcium lost in the breast milk (242).

The skeletal losses are due in part to the low estradiol levels during lactation which stimulate osteoclast number and activity. However, low estradiol is not the sole cause of the accelerated bone resorption or other changes in calcium homeostasis that occur during lactation. It is worth noting what happens to reproductive-age women who have marked estrogen deficiency induced by GnRH agonist therapy in order to treat endometriosis, fibroids, or severe acne. Six months of GnRH-induced estrogen deficiency caused 1-4% losses in trabecular (but not cortical) aBMD, increased urinary calcium excretion, and suppression of calcitriol and PTH (Figure 4) [reviewed in (1,8)]. In contrast, during lactation women are not as estrogen deficient but lose more aBMD (at both trabecular and cortical sites), have normal (as opposed to low) calcitriol levels, and have reduced (as opposed to increased) urinary calcium excretion (Figure 4). The difference between isolated GnRH-induced estrogen deficiency and lactation appears to be explained by PTHrP. It stimulates osteoclast-mediated bone resorption and stimulates renal calcium reabsorption; by so doing, it complements the effects of low estradiol during lactation. Stimulated in part by suckling and high prolactin levels, PTHrP and estrogen deficiency combine to cause marked skeletal resorption during lactation (Figure 5).

Figure 4. Comparison of the effects of acute estrogen deficiency vs. lactation on calcium and bone metabolism. Acute estrogen deficiency (e.g. GnRH analog therapy) increases skeletal resorption and raises the blood calcium; in turn, PTH is suppressed and renal calcium losses are increased. During lactation, the combined effects of PTHrP (secreted by the breast) and estrogen deficiency increase skeletal resorption, reduce renal calcium losses, and raise the blood calcium, but calcium is directed into breast milk. Reprinted from ref. (8), © 1997, The Endocrine Society.

Figure 5. Brain-Breast-Bone Circuit. The breast is a central regulator of skeletal demineralization during lactation. Suckling and prolactin both inhibit the hypothalamic gonadotropin-releasing hormone (GnRH) pulse center, which in turn suppresses the gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), leading to low levels of the ovarian sex steroids (estradiol and progesterone). PTHrP production and release from the breast is controlled by several factors, including suckling, prolactin, and the calcium receptor. PTHrP enters the bloodstream and combines with systemically low estradiol levels to markedly upregulate bone resorption. Increased bone resorption releases calcium and phosphate into the blood stream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into milk at high concentrations, but whether swallowed PTHrP plays a role in regulating calcium physiology of the neonate is unknown. Calcitonin (CT) may inhibit skeletal responsiveness to PTHrP and low estradiol. Not depicted are that direct effects of oxytocin and prolactin on bone cells are also possible. Adapted from ref. (26) © 2006, The Endocrine Society.

The mechanism through which the skeleton is resorbed has been shown in rodents to involve two processes, both osteoclast-mediated bone resorption (1) and osteocytic osteolysis, in which osteocytes function like osteoclasts to resorb the bone matrix that surrounds them (243). Both of these processes are dependent upon PTHrP. Conditional deletion of the PTHrP gene from mammary tissue reduced the amount of bone resorbed during lactation, whereas conditional deletion of the PTH/PTHrP receptor from osteocytes appeared to eliminate osteocytic osteolysis (244). Moreover, osteocyte-specific deletion of the PTH/PTHrP receptor resulted in a 50% blunting of the amount of aBMD lost during lactation (244), which may indicate that osteocytic osteolysis and osteoclast-mediated bone resorption each contribute about half of the net bone loss achieved during lactation. To date no studies have examined whether osteocytic osteolysis occurs in lactating women.

The lactational bone density losses in women are substantially and completely reversed during six to twelve months following weaning (1,60,239). This corresponds to a gain in bone density of 0.5 to 2% per month in a woman who has weaned her infant. The mechanism for this restoration of bone density is unknown, but studies in mice have shown that it is not dependent upon calcitriol, calcitonin, PTH, or PTHrP (17,23,26,47,245,246); nor is it fully explained by restoration of estradiol levels to normal (1). The remarkable ability of the skeleton to recover is exemplified by mice lacking the gene that encodes calcitonin. They lose up to 55% of trabecular mineral content from the spine during lactation but completely restore it within 18 days after weaning (26).

Although aBMD appears to be completely restored after weaning in women and all animals that have been studied, more detailed examination of microarchitecture by µCT has shown variable completeness of recovery of microarchitecture by skeletal site. In rodents, the vertebrae recover completely while persistent loss of trabeculae is evident in the long bones (247). Studies in women have similarly shown that the trabecular content of the long bones also appears to be incompletely restored (1,60,239,248,249). However, in both women (77,249,250) and rodents (26,251,252) the cross-sectional diameters and volumes of the long bones may be significantly increased after post-weaning. Such structural changes potentially compensate for any reduction in strength that loss of trabecular microarchitecture might induce, because an increased cross-sectional diameter increases the ability of a hollow shaft to resist bending (cross-sectional moment of inertia) and torsional stress (polar moment of inertia). This is supported by the finding that the breaking strength of rodent bones returns to pre-pregnant values after weaning (1,245), and limited clinical studies that correlated the increased bone volumes achieved after reproductive cycles with increased bone strength (77,250). In women, the vast majority of several dozen epidemiologic studies of pre- and postmenopausal women have found no adverse effect of a history of lactation on peak bone mass, bone density, or hip fracture risk (1,7,57,60). In fact, multiple studies have suggested a protective effect of lactation on the future risk of low aBMD or fragility fractures. Consequently, although lactational bone loss can transiently increase risk of fracture (see next section), it is likely unimportant in the long run for most women, in whom the skeleton is restored to its prior mineral content and strength.

DISORDERS OF CALCIUM AND BONE METABOLISM DURING LACTATION

Osteoporosis of Lactation

On occasion a woman will suffer one or more fragility fractures during lactation, and osteoporotic bone density will be found by DXA (79). As with osteoporosis presenting during pregnancy, this may represent a coincidental, unrelated disease; the woman may have had low bone density and abnormal skeletal microarchitecture prior to pregnancy, such that the normal bone loss incurred by lactation could not be tolerated. Alternatively, it is likely that some cases represent an exacerbation of the normal degree of skeletal demineralization that occurs during lactation, and a continuum from the changes in bone density and bone turnover that occurred during pregnancy. For example, the skeleton may have been normal pre-pregnancy, lost mineral content during pregnancy due to low calcium intake, and experienced the expected further loss during lactation (79). It may be somewhat artificial, therefore, to separate “osteoporosis of lactation” from “osteoporosis of pregnancy.” But since lactation normally causes a significant net loss of bone whereas pregnancy does not, it seems more likely for lactation to cause a subset of women to develop low-trauma fractures. For example, excessive PTHrP release from the lactating breast into the maternal circulation could conceivably cause excessive bone resorption, osteoporosis, and fractures. PTHrP levels were high in one case of lactational osteoporosis, and remained elevated for months after weaning (253).

The literature can be confusing because “pregnancy-associated osteoporosis” is the term often used for bone loss and fractures that present during or after pregnancy, despite such fractures being more likely to have resulted from the bone loss during lactation. In fact, multiple case series have demonstrated that about 80-90% of the fragility fractures associated with reproductive cycles occur during lactation, with the remaining 10-20% occurring either during pregnancy or in the puerperium for women who do not breastfeed. Therefore, the term “pregnancy and lactation-associated osteoporosis” (PLO) is a more suitable one to use.

The earlier, longer discussion about osteoporosis of pregnancy should be reviewed for more details since everything in that section applies to osteoporosis presenting during lactation. The skeleton may be normal or abnormal prior to pregnancy, bone loss may have occurred during pregnancy, and bone loss will certainly occur during lactation. The magnitude of bone loss during lactation correlates with the volume of breast milk produced, which in turn correlates with the duration of near-exclusive or exclusive breast feeding (i.e., that most or all of the baby’s nutrition comes from breast milk).

TREATMENT CONSIDERATIONS

The diagnostic and treatment considerations described earlier for osteoporosis of pregnancy also apply to women who are lactating (79). Case series have revealed that a spontaneous 20-70% increase in bone density occurs in women who fractured while breastfeeding (1,79,80,85,86,104-112). Therefore, pharmacological therapy may be best avoided for 12 to 18 months to determine the extent of spontaneous recovery, and then decide if additional treatment is necessary (79,86). The extent of spontaneous recovery of lumbar spine aBMD at 12–18 months should be assessed by DXA. As noted in the pregnancy section, HR-pQCT will underestimate the extent of recovery at this early stage unless the parameters are adjusted to detect under-mineralized bone and osteoid.

The mechanism through which post-weaning recovery occurs is not established, but a theoretical concern is that anti-remodeling agents such as bisphosphonates or denosumab might blunt spontaneous recovery since they suppress bone formation. Furthermore, none of the available pharmacotherapies are indicated for use in premenopausal women, and especially not in women who continue to breastfeed. As noted in the earlier section on osteoporosis associated with pregnancy, individual case reports and series have described marked increases in bone mass in association with pharmacotherapy use after lactation, but in each of these cases the magnitude of increases were in keeping with that achieved through spontaneous recovery (86). A few reports compared to women treated with vitamin D and calcium alone, and there was no difference in the final aBMD achieved with pharmacotherapy (usually teriparatide or bisphosphonates) vs. spontaneous recovery (83,109,119,120). One large case series of 107 women found that subsequent fractures were twice as likely to occur in women who had received pharmacotherapy, which suggests that pharmacotherapy may lead to weaker bone (109). Consequently, it is unclear whether early use of pharmacotherapy after lactation-induced bone loss achieved any added benefit.

Primary Hyperparathyroidism

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

TREATMENT CONSIDERATIONS

The albumin-corrected serum calcium may subside after pregnancy due to intestinal calcium absorption returning to normal after the pregnancy-induced increase. Consequently, there may be no urgency for parathyroidectomy to be done unless a parathyroid crisis occurs. If future pregnancies are planned, then it will be prudent for a neck exploration to be done to correct the condition in advance of another pregnancy. Breastfeeding may require that surgery and required localization procedures be delayed.

Familial Hypocalciuric Hypercalcemia

The calcium-sensing receptor is expressed in mammary epithelial ducts, and it modulates the production of PTHrP and calcium transport into milk during lactation in mice (254,255). Inactivating calcium-sensing receptor mutations increased mammary tissue production of PTHrP but decreased the calcium content of milk (255). These opposing changes meant that there was a further increase in bone resorption during lactation as compared to normal mice, and the serum calcium also became higher because of reduced output of calcium into milk. Conversely, a calcimimetic drug (similar to cinacalcet) caused increased milk calcium content (255). These data predict that women with FHH will have more marked skeletal resorption during lactation, lower milk calcium content, higher serum calcium, and a greater loss of aBMD during lactation as compared to normal women. However, the effect of breastfeeding on mineral and skeletal homeostasis in women with FHH has not yet been described.

TREATMENT CONSIDERATIONS

Women with FHH can be expected to breastfeed normally and do not require any treatment. It would be of interest for observational studies to be done to clarify if they experience excess bone loss and produce milk with lower calcium content, as compared to women without FHH.

Hypoparathyroidism

As noted earlier, in the first day or two after parturition the requirement for supplemental calcium and calcitriol may transiently increase in hypoparathyroid women before secretion of PTHrP surges in the breast tissue (159). The onset of lactation induces an important change in skeletal metabolism because the breasts produce PTHrP at high levels, some of which escapes into the maternal circulation to stimulate bone resorption and raise the serum calcium level. In women who lack parathyroid glands, the release of PTHrP into the circulation during lactation can temporarily restore calcium and bone homeostasis to normal. Levels of calcitriol and calcium supplementation required for treatment of hypoparathyroid women fall early and markedly after the onset of lactation, and hypercalcemia can occur if the calcitriol dosage and calcium intake are not substantially reduced (256-259). This decreased need for calcium and calcitriol occurs at a time when circulating PTHrP levels are high in the maternal circulation (256,259,260). As illustrated in one case, this is consistent with PTHrP reaching the maternal circulation in amounts sufficient to allow stimulation of calcitriol synthesis, and maintenance of normal (or slightly increased) maternal serum calcium (260).

TREATMENT CONSIDERATIONS

Management of hypoparathyroidism during lactation requires monitoring the albumin-corrected calcium or ionized calcium, reducing or stopping the calcitriol and calcium as indicated, and planning to reinstitute both supplements in escalating doses as lactation wanes. However, production of PTHrP doesn’t necessarily promptly cease around the time of weaning. The author is aware of a woman with hypoparathyroidism who required no supplemental calcium or calcitriol at all for about a year after her baby had been weaned. She thought that her hypoparathyroidism had been permanently cured by breastfeeding, until the abrupt recurrence of symptomatic hypocalcemia, and the need for pre-pregnancy doses of calcium and calcitriol, signaled the end of PTHrP production by her breasts. In another woman, lactation appeared to permanently cure her hypoparathyroidism (261), likely because of persistent production of PTHrP by her breasts.

Pseudohypoparathyroidism

The management of pseudohypoparathyroidism during lactation has been less well documented. Since these patients are likely resistant to the renal actions of PTHrP, and the placental sources of calcitriol are lost at parturition, the calcitriol requirements might well increase and may require further adjustments during lactation. Conversely, these patients do not have skeletal resistance to PTH, and so it is possible that calcium and calcitriol requirements may decrease secondary to enhanced skeletal resorption caused by the combined effects of high PTH levels, PTHrP release from the breast, and lactation-induced estrogen deficiency. Thus, women with pseudohypoparathyroidism might lose more bone density than normal during lactation, but this has not been studied.

TREATMENT CONSIDERATIONS

In the absence of data, it would be best to monitor the albumin-corrected serum calcium to determine if any adjustments are needed in the doses of oral calcium and calcitriol.

Pseudohyperparathyroidism

Severe, PTHrP-mediated hypercalcemia during lactation was first noted to occur in women with large breasts, but it has also developed in women with average-sized breasts in whom milk let-down took place but the baby’s illness prevented breastfeeding (176). This represents an exaggeration of normal lactational physiology, which benefits hypoparathyroid women, but in some normal women can overwhelm the normal regulatory pathways and cause potentially severe hypercalcemia.

TREATMENT CONSIDERATIONS

Cessation of lactation should reverse the condition, aided by use of breast-binders, and bromocriptine or cabergoline to suppress prolactin. However, a reduction mammoplasty or mastectomy has proved necessary for recalcitrant hypercalcemia in some cases.

Vitamin D Deficiency and Insufficiency, and Genetic Vitamin D Disorders

The mother andata from small clinical trials, observational studies and case reports indicate that lactation proceeds normally regardless of vitamin D status, and breast milk calcium content is unaffected by vitamin D deficiency or supplementation in doses as high as 6,400 IU per day given to the mother, which achieved maternal 25OHD blood levels of 168 nmol/L (topic reviewed in detail in (1,4,5,7,179)). This is likely because maternal calcium homeostasis is dominated by skeletal resorption induced by estrogen deficiency and PTHrP, with vitamin D/calcitriol playing no substantial role in lactational mineral homeostasis. It is the neonate who will suffer the consequences of being born of a vitamin D deficient mother. This is especially true if the infant is exclusively breast fed, since both vitamin D and 25-hyroxyvitamin D are normally present at very low concentrations in breast milk.

The high-dose (6,400 IU) vitamin D supplementation strategy raises the maternal vitamin D concentration substantially for hours and, in turn, this increases the penetration of vitamin D into milk. Consequently, breastfed babies whose mothers consumed 6,400 IU per day achieved the same 25OHD level as babies who received a 300 IU dose of vitamin D directly (262). The potential advantage of this approach is that all of the neonate’s nutrition can then come from breast milk, rather than requiring that breastfed babies receive a vitamin D supplement. Further study is needed regarding the safety of this approach for the mothers and their babies. A Cochrane review also concluded there is no established benefit from high-dose supplementation of breastfeeding women, as compared to the normal route of giving a lower dose of vitamin D directly to a breastfed baby (263).

A misconception about vitamin D and milk often arises because marketed forms of cow’s and goat’s milk contain approximately 100 IU of vitamin D per standard serving, but that is a synthetic vitamin D supplement which is added to the milk after the pasteurization stage. It is not put there by the cow or goat.

Given that vitamin D deficiency does not affect breast milk content in humans, it is likely that genetic absence of VDR or calcitriol also does not affect milk calcium, but this has not been studied.

Whether vitamin D deficiency impairs the ability of the maternal skeleton to recover post-weaning has not been examined in any clinical study. However, studies in mice lacking the vitamin D receptor or Cyp27b1 to synthesize calcitriol, indicate that these mice are able to fully remineralize their skeletons after lactation (23,47).

TREATMENT CONSIDERATIONS

Breastfeeding women have the same vitamin D intake requirements as non-pregnant and pregnant women (221). Therefore, no change in dose of any oral supplements is needed. Penetrance of vitamin D into breast milk is poor, and so breastfed babies need oral vitamin D supplementation to prevent vitamin D deficiency, until such time as vitamin D-supplemented infant nutrition is taken.

24-Hydroxylase Deficiency

Hereditary absence of Cyp24a1 reduces calcitriol catabolism, which can lead to very high calcitriol concentrations and marked maternal hypercalcemia during pregnancy. But calcitriol production falls to non-pregnant levels during normal lactation, and the same should be true in women with 24-hydroxylase deficiency. Consistent with this, in one affected woman who breastfed, calcitriol was normal and hypercalcemia was milder compared to pregnancy (205).

TREATMENT CONSIDERATIONS

Breastfeeding appears unlikely to require any intervention in women with 24-hydroxylase deficiency.

Low and High Calcium Intakes

The calcium content of milk appears to be largely derived from skeletal resorption during lactation, a process that cannot be suppressed in women by consuming greater amounts of calcium (however, it can be suppressed in rodents by high calcium intakes). It shouldn’t be surprising, therefore, that low calcium intake does not impair breast milk quality, nor does it accentuate maternal bone loss (216). Even in women with very low calcium intakes, the same amount of mineral was lost during lactation from the skeleton as compared to women who had supplemented calcium intakes, and the breast milk calcium content was unaffected by calcium intake or vitamin D status (264-266). Conversely, since randomized trials and cohort studies have shown that high calcium intakes do not affect the degree of skeletal demineralization that occurs during lactation (238-241), it is unlikely that increasing calcium supplementation well above normal would affect skeletal demineralization either.

There is a lingering concern that adolescent mothers with low calcium intakes may not achieve normal peak bone mass as a consequence of lactation-induced bone loss. In fact the adolescent skeleton appears to recover fully from lactation (267), and adolescent women who breastfed have higher aBMD than those who did not breastfeed or had not been pregnant as adolescents (268). However, it remains reasonable to give a calcium supplement to adolescents who lactate in order to ensure that the needs of adolescent growth are met and that peak bone mass is achieved (216,267).

TREATMENT CONSIDERATIONS

A normal calcium intake of 1,000-1,200 mg daily, as suggested by the Institute of Medicine (221), is recommended for breastfeeding women. Low and high calcium intake should be avoided. It is well established that breastfeeding women do not require increased calcium intake.

FGF23 Disorders

There is very limited information available about the effect of FGF23-related disorders on mineral homeostasis during lactation, and milk production. Lactation normally causes the serum phosphorus to rise due to the increased release of phosphate from the resorbing skeleton. Indeed in one case report, serum phosphorus increased into the normal range in a breastfeeding woman with XLH (223). Curiously, the phosphate content of expressed milk was reduced to 50% of normal in two cases of XLH (223,224), which differs from findings in animal models of XLH in which milk phosphate content was normal (269). Use of oral phosphate supplementation normalized the milk phosphate content in one case where this intervention was studied (223).

Milk phosphate content has not been studied in disorders in which FGF23 activity is reduced; however, given the findings with XLH, it is conceivable that milk phosphate content will be increased.

TREATMENT CONSIDERATIONS

The mineral content of milk is not normally analyzed. Given the limited findings from two cases of XLH, it may be prudent to recommend that oral phosphate supplementation be maintained while breastfeeding, even if the serum phosphorus has spontaneously normalized. If the baby develops hypophosphatemia, this may indicate inadequate milk phosphate content or that the baby inherited XLH. Use of burosumab has not been described in breastfeeding women, but this large protein should have low penetrance into milk and is likely to be destroyed in the infant’s gastrointestinal tract (270).

IMPLICATIONS

During pregnancy and lactation, novel regulatory systems specific to these settings complement the usual regulators of mineral homeostasis. Intestinal calcium absorption more than doubles from early in pregnancy in order to meet the fetal demand for calcium. In comparison, skeletal calcium resorption is a dominant mechanism by which calcium is supplied to the breast milk, while renal calcium conservation is also apparent. Calcium supplementation during pregnancy will result in a woman absorbing more calcium, but it is clear from clinical trials and observational studies that calcium supplements have little or no impact on the amount of bone lost during lactation.

The skeleton appears to recover promptly from lactation to achieve the pre-pregnancy bone mass through mechanisms that remain unclear. The transient loss of bone mass during lactation can at least temporarily compromise skeletal strength and rarely lead to fragility fractures. Furthermore, full recovery of mineral content and bone strength may not always be achieved after weaning. But the majority of women can be assured that the changes in calcium and bone metabolism during pregnancy and lactation are normal, healthy, temporary, and without adverse consequences in the long-term.

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Empty Sella

ABSTRACT

Empty sella is a radiological finding of a flattened pituitary in a sellar space filled with cerebrospinal fluid. It may be primary or secondary consequent to various processes causing injury and shrinkage of the pituitary gland (postpartum hemorrhage, pituitary surgery, irradiation, apoplexy, infection, head trauma, hypophysitis, etc.). The mechanisms involved in the pathogenesis of the so called “primary empty sella” may range from continuously or intermittently increased intracranial pressure due to idiopathic benign intracranial hypertension, obesity, arterial hypertension, or multiple pregnancies in female patients with accompanying insufficiency of the sellar diaphragm and changes in pituitary gland volume (hyperplasia during pregnancy, lactation, menopause etc.). Primary empty sella can be an incidental radiological finding in an asymptomatic patient with preserved pituitary function. In symptomatic patients with the so called “empty sella syndrome” (headache, visual disturbances, and hormonal dysfunction), the radiological finding of an empty sella is important in the differential diagnosis of other sellar lesions. Hypopituitarism, partial or complete, and hyperprolactinemia are not uncommon in these patients. The treatment of hypopituitarism and hyperprolactinemia is advocated in all patients with confirmatory results. In patients with the secondary empty sella, hypopituitarism is more common and more readily recognized due to damage caused by surgery, radiation therapy, or various pathological causes. Rarely, an empty sella can be associated with hormonal hypersecretion from an “invisible” micro adenoma producing prolactin, growth hormone (acromegaly), or ACTH (Cushing’s disease). A wide range of radiological findings in patients with secondary and primary empty sella coupled with clinical data (important hints from the history and data on endocrine function) are presented for further illustration of this topic.

HISTORY

The term ‘empty sella’ was first used by Bush in 1951 (1) to describe a peculiar anatomical condition, observed in 40 of 788 human cadavers, particularly females, characterized by a sella turcica with an incomplete diaphragm sellae that forms only a small peripheral rim, with a pituitary gland not absent, but flattened in such a manner as to form a thin layer of tissue at the bottom of the sella turcica. Kaufman (2), in 1968, speculated that ‘…empty sella is a distinct anatomical and radiographic entity, function of an incompleteness of the diaphragma sellae and of the cerebrospinal fluid (CSF) pressure, normal or elevated’. The role of the normal fluctuations of CSF pressures and the effect of a superimposed prolonged increase in CSF pressure were related to the anatomic changes involving the bony wall of the empty sella.

DEFINITION, ETIOLOGY, AND PREVALENCE OF EMPTY SELLA

Empty sella is defined as herniation of subarachnoid space into the sella turcica (arachnoidocele). It is a term for the radiological finding of “empty sellar space” on magnetic resonance imaging (MRI) and computerized tomography (CT) with a flattened pituitary and elongated stalk. It can be partial if less than 50% of sellar space is filled with cerebro-spinal fluid (CSF), or complete if CSF fills more than 50% of space in the sella and gland thickness is less than 2mm (3,4).

Regarding its etiology, it can be primary if there is no pathological process in the sellar region preceding the pituitary damage or secondary if it is consequent to a specific pathological process. Primary empty sella (PES) can be an incidental finding or may arise during imaging for headache, endocrine disorders, neurological symptoms, visual disturbances, abnormal sella turcica radiograph, and other reasons.

Primary empty sella (PES) can be caused by intracranial hypertension and/or insufficiency of the sellar diaphragm in subjects with no previous history of pituitary disease. Insufficiency of the sellar diaphragm, a deflection of dura matter separating the suprasellar cistern from the pituitary fossa, allows unobstructed pulsatile movements of CSF from chiasmatic cistern causing flattening of the pituitary to the sellar floor. In extreme cases bone erosion of the sellar floor and CSF leak (rhinorrhea) may occur, increasing the risk of meningitis. Partial or complete absence of the sellar diaphragm has been demonstrated in patients with PES.

Intracranial pressure can be intermittently increased due to obesity, sleep apnea, arterial hypertension, pregnancy, and labor. Intracranial hypertension may be idiopathic or associated with other intracranial processes such as tumors, venous thrombosis, infections, or malformations. Idiopathic intracranial hypertension (IIH) or “pseudo-tumor cerebri” is a rare condition affecting 1 in 100,000 persons. It can be due to impaired CSF absorption, increased CSF secretion, and/or increased capillary permeability (5). Impaired CSF dynamics and absorption have been found in up to 77% and 84% of patients with PES, respectively (6), The prevalence of PES is very high in patients with IIH ranging from 70-94% (4).

Changes in the pituitary gland volume may also be involved in the pathogenesis of empty sella syndrome including hyperplasia during pregnancy and lactation and pituitary involution after menopause accounting for the significantly higher prevalence of this condition in female patients (female to male ratio 5:1).

Factors involved in the pathogenesis of primary empty sella are shown in Figure 1.

Secondary empty sella is more common and is related to various pathological processes of the sellar region. Among many causes, pituitary tumor shrinkage occurring after medical treatment, surgery, radiotherapy, and apoplexy of a pituitary adenoma are frequent causes of secondary empty sella. Likewise, postpartum pituitary necrosis, pituitary infection, hypophysitis, and traumatic brain injury may lead to pituitary atrophy. The diagnosis of secondary empty sella is more difficult if there is no known underlying pathology involving the pituitary gland. In these cases, the sella is normal in size, and the function of the flattened pituitary gland may or may not be compromised. Such is the case in congenital causes of hypopituitarism both acquired and genetic, presenting with a hypoplastic pituitary gland and ectopic posterior lobe (7). Large intracranial tumors such as slow-growing meningiomas can also cause increased intracranial pressure and secondary empty sella in a significant number of patients. Pituitary MRI images of patients with secondary and primary empty sella are presented in the section dedicated to radiological appearance of an empty sella (Fig. 2-6). Associated risk factors for primary and secondary empty sella are shown in Table 1.

The reported prevalence of empty sella depends on techniques used for detection. In autopsy studies empty sella has been found in up to 5.5-12% cases (1,2), On imaging the overall incidence has been estimated at 12% (4). The prevalence of PES is very high in patients with IIH (8)

Table 1. Associated Risk Factors for Primary and Secondary Empty Sella
Primary Empty Sella (PES)	Secondary Empty Sella (SES)
Female sex Multiple pregnancies Obesity and sleep apnea Arterial hypertension Benign intracranial hypertension	Medical therapy Pituitary surgery Irradiation Pituitary apoplexy Sheehan’s syndrome Traumatic brain injury Congenital hypopituitarism

Table 1. Associated Risk Factors for Primary and Secondary Empty Sella

Primary Empty Sella (PES)

Secondary Empty Sella (SES)

Female sex

Multiple pregnancies

Obesity and sleep apnea

Arterial hypertension

Benign intracranial hypertension

Medical therapy

Pituitary surgery

Irradiation

Pituitary apoplexy

Sheehan’s syndrome

Traumatic brain injury

Congenital hypopituitarism

Figure 1. Factors involved in pathogenesis of primary empty sella include the upper-sellar factors, incompetence or incomplete formation of the sellar diaphragm, and pituitary factors associated with the variation in the pituitary volume. Modified from Bioscientifica Ltd., Chiloiro S et al, European Journal of Endocrinology (2017) 177, R275–R285

RADIOLOGICAL APPEARANCE OF EMPTY SELLA

Very commonly an empty sella is incidentally discovered during MRI or CT imaging or during evaluation for headache, endocrine, neurological or visual disturbances. Less commonly it is observed after additional imaging for abnormal sella turcica radiographs. Chronic intracranial hypertension can lead to sellar remodeling and enlargement with thinning of the sellar floor and rarely rhinorrhea.

On typical presentation CSF filling is in continuity with overlying subarachnoid spaces and the residual pituitary gland is flattened against the sellar floor of an enlarged bony sella with pituitary volume usually less than 611.21 mm³ (9). The differential diagnosis with cystic lesions and congenital pituitary abnormalities may pose a challenge. The stalk is usually thinned and located in the midline. Asymmetry is a frequent sign of the secondary empty sella. In rare cases the chiasm can herniate into the sella in cases of both primary and secondary etiology. Indirect signs of intracranial hypertension may also be present such as flattening of the posterior sclera, prominent subarachnoid spaces along the optic nerves, vertical tortuosity of the optic nerve sheath complex, and increased width of the optic nerve sheaths (10).

Sagittal and coronal T1-weighted (T1W) contrast enhanced images and coronal T2-weighted (T2W) images are strongly indicated for MR studies because they show CSF within the sella. On FLAIR sequences the intrasellar fluid completely suppresses and it presents without restriction in DWI sequences. After contrast T1W images show normal enhancement of residual pituitary gland and the stalk in PES in contrast to scaring and distortion in SES.

In patients with congenital hypopituitarism an ectopic posterior pituitary, stalk duplication or absence may be present in combination with other midline defects (agenesis of corpus callosum, supraoptic dysplasia, etc.) (7).

Figures 2, 3, 4, 5 represent various MRI findings of patients with secondary empty sella due to postpartum hemorrhage (Sheehan’s syndrome Fig. 2), lymphocytic hypophysitis (Fig. 3), shrinkage of a macroprolactinoma after successful treatment with cabergoline (Fig.4), and congenital hypopituitarism with empty sella (Fig.5)

Figure 2. Sagittal T1W images of two patients with empty sella and Sheehan’s syndrome
Left Panel (sagittal T1W image without contrast) and Right (sagittal T1W image after contrast enhancement) represent the MRI appearance of empty sella in two patients with Sheehan’s syndrome. Both patients presented with hyponatremic coma due to unrecognized panhypopituitarism and infection 3 and 20 years after delivery complicated by postpartum hemorrhage.

Figure 3. Contrast enhanced coronal and sagittal T1W images of lymphocytic hypophysitis spontaneous evolution from the presentation (panel A, B), after 4 (panel C) and 10 years (panel D) of follow-up resulting in secondary empty sella.

Figure 4. Coronal T1W images demonstrating a pituitary tumor (macroprolactinoma) shrinkage in a patient treated with the dopamine agonist cabergoline for 10 years. The patient presented with hyperprolactinemia causing galactorrhea-amenorrhea, secondary adrenal insufficiency and central hypothyroidism. Hyperprolactinemia and adrenal insufficiency completely recovered during follow up.

Figure 5. Sagittal T1W showing small pituitary gland found at the bottom of sella, thin stalk and ectopic posterior lobe in a young adult with isolated childhood-onset growth hormone deficiency (congenital).

Figure 6. Sagittal T1W images of pituitary stalk pressed against the dorsum sellae causing mild hyperprolactinemia in a patient with primary empty sella.

PRIMARY EMPTY SELLA (PES) AND EMPTY SELLA SYNDROME

Epidemiologically, this is associated with female sex (female to male ratio 5:1), multiple pregnancies, obesity, arterial hypertension, and middle age. It may present with headaches, endocrine dysfunction, and visual disturbances due to pressure on the neighboring structures.

A typical clinical picture consists of headache and obesity. Women in the reproductive age may be affected by menstrual irregularities, galactorrhea, and sterility. Man can develop gynecomastia and sexual disturbances. Primary empty sella due to the syndrome of increased intracranial pressure can also be associated with symptoms of intracranial hypertension.

Pathogenesis of Primary Empty Sella (PES)

Pregnancy may trigger the onset of PES. It is associated with pituitary hyperplasia and CSF hypertension especially in multiple pregnancies. PES has also been associated with CSF hypertension related to obesity and arterial hypertension. In the largest study with 175 patients, multiple pregnancies were reported in 58.3% women with PES, while obesity and arterial hypertension were recorded in 49.5% and 27.3% of patients (10). In patients with benign intracranial hypertension, empty sella is a common finding (8, 11).

Endocrine Dysfunction in PES

Hyperprolactinemia, usually mild (less than 50 ng/ml), is present in approximately 10% of patients (10). It is often due to increased pressure of the CSF on the pituitary stalk and diminished dopamine inhibitory effect. Prolactin dynamics in PES may be influenced by gonadal status, intracranial pressure, neurotransmitters, and stalk integrity. Rarely, pituitary microadenomas causing acromegaly and Cushing’s disease may be associated with empty sella. In the recent study by Himes et al. empty sella was associated with an increased rate of MRI negative Cushing’s disease (12). Pituitary compression causing a relative reduction in the volume of the pituitary for imaging is a plausible cause for not detecting the tumor mass with MRI (12). The presence of ES was associated with lower preoperative prolactin and nadir GH responses to OGTT in patients with acromegaly (13) Functional imaging, if available may identify occult pituitary tumors as therapeutic targets in such settings.

The prevalence of hypopituitarism in patients with PES is variable. In a study by Guitelman et al. it was found in 28% of patients (11). Panhypopituitarism was present in 40% of these patients, while partial or isolated hormone deficiencies were diagnosed in 60% of hypopituitary patients (11). The most prevalent pituitary deficiency was growth hormone deficiency.

In a pooled meta-analysis, which included four studies, the frequency of hypopituitarism was 52% (14). Multiple pituitary hormone deficiencies were present in 30%, isolated in 21% of patients with PES. Growth hormone and gonadotropins were the most common isolated insufficiencies (14). Significant correlation was reported between IGF-1 values and pituitary volume measurements (15).

In a retrospective single-center study of 765 patients by Ekhzaimy et al. 79% of PES was diagnosed incidentally on MRI. The majority of patients were evaluated by general practitioners with suboptimal hormonal evaluation while only 20 % were referred to endocrinologists for hormonal evaluation (16).

A multicenter retrospective study from Italy, detected hormonal alterations in 29% of incidental PES suggesting the need for careful initial evaluation since on follow-up hormonal deterioration was uncommon (3%) (17). Hypopituitarism was associated with male sex (17, 18) and radiological finding of complete PES (18).

Hormonal assessment is advocated in all patients with ES. In case of borderline results or suspected isolated or partial insufficiency stimulatory tests are recommended if clinically relevant for hormone replacement.

Other symptoms in patients with ES include headache and visual and neurological disturbances. In patients with intracranial hypertension these symptoms are more common.

Neurological and Ophthalmic Dysfunction in PES

Headache is present in approximately 80% (3,6). In 20% of patients it may be accompanied by visual disturbances, even papilledema in intracranial hypertension (3,6).

Visual disturbances including worsening of visual acuity, blurred vision, diplopia, defects of oculomotor nerve, and optical neuritis were also reported in patients with PES. In case of benign intracranial hypertension ophthalmic echography and computerized visual field with evoked potentials should be performed in consultation with ophthalmologists. Study by Yilmaz et al. evaluated retinal optic nerve fiber thickness by optic coherence tomography in asymptomatic patients with empty sella and healthy controls (19). Reduced values in asymptomatic patients provide valuable data for monitoring of these patients (19). In case of chiasmal herniation into the empty sella with acute visual deterioration, new neurosurgical techniques of chiasmal transsphenoidal elevation are available (20).

Neurological disturbances including dizziness, syncope, cranial nerve disorders, convulsions, and depression were reported in approximately 40% of patients with PES (3, 6). Rhinorrhea increases the risk of meningitis and surgery involving osseous remodeling techniques (21).

An association of PES with periventricular white matter hyperintensities, and enlarged perivascular spaces, common features of cerebral small vessel disease, were reported confirming the previous clinical observation and possible common underlying mechanism (22).

TREATMENT

In patients with increased idiopathic intracranial pressure osmotic diuretics or acetazolamide (Diamox) are advocated. Weight loss may be helpful in obese and overweight patients especially if accompanied by sleep apnea. Neurosurgical techniques may be indicated for rhinorrhea and some symptomatic secondary causes of empty sella syndrome with increased intracranial pressure and acute visual disturbances. If hypopituitarism is present it should be treated following the current recommendations (23) and hyperprolactinemia treated with dopamine agonists.

CONCLUSION

Primary empty sella can be heterogeneous in origin and presentations range from an asymptomatic incidental radiological finding to endocrine and neuro-ophthalmological manifestations. Female sex, multiple pregnancies, obesity, and arterial hypertension are associated risk factors as well as the syndrome of benign intracranial hypertension. Secondary empty sella is caused by various pathological processes resulting in shrinkage of the pituitary gland. Routine hormonal status assessment and regular follow-up are indicated in all patients since the prevalence of pituitary dysfunction is significant.

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Adrenal Insufficiency In Children

ABSTRACT

Adrenal insufficiency (AI) is an uncommon but potentially life-threatening condition related to impaired secretion of cortisol by the adrenal gland. In general, this condition can be divided into primary (adrenal failure) and central (hypothalamic/pituitary) causes. In this chapter, we categorize the causes of adrenal insufficiency and systematically review the etiologies and associations to guide the laboratory evaluation and treatment, specifically as it pertains to the pediatric patient. Early diagnosis and treatment can prevent the development of adrenal crisis – prompt recognition can be lifesaving. Understanding the manifestation of unique types of adrenal insufficiency can guide management with glucocorticoid +/- mineralocorticoids and guide further investigation for associated disorders. We discuss the treatment of adrenal insufficiency, reviewing both the acute (crisis) and chronic management.

INTRODUCTION

Adrenal insufficiency (AI) refers to impaired cortisol secretion by the adrenal gland. If untreated, AI can be life-threatening, especially when it is compounded by a physiological stress, such as an acute illness, severe trauma, or surgical procedure (1,2).

Cortisol secretion is regulated by the hypothalamic-pituitary-adrenal axis (HPA) (Figure 1) (1,2). In brief, Corticotropin-releasing hormone (CRH) secreted by the paraventricular nuclei of the hypothalamus stimulates adrenocorticotropic hormone (ACTH) production and release by the pituitary, leading to cortisol synthesis and secretion by the adrenal gland. Cortisol then negatively feeds back to inhibit both CRH and ACTH release. Clinical manifestations of this feedback mechanism occur frequently as illustrated in clinical practice after chronic treatment with exogenous glucocorticoids, which may suppress CRH and ACTH secretion and consequently result in adrenal atrophy and AI. In addition, a high day (light) and low night (dark) diurnal cycle of ACTH release is entrained, permitting optimal times for assessing the status of spontaneous cortisol secretion (early morning) or its optimal suppression (around midnight).

The fetal adrenal gland markedly differs in structure and function from that of the adult (3,4). During both early and late gestation, the adrenal cortex consists primarily of the fetal zone, which produces predominantly androgens that are critical for the sexual maturation of fetal external genitalia. Early cortisol production is indirect via placental conversion of progesterone synthesized in the fetal adrenal to cortisol, whereas, toward the end of the 2^nd trimester, direct cortisol synthesis occurs in the definitive zone of the fetal adrenal (5). Aldosterone synthesis remains low until the end of gestation at which time CYP11B2 expression begins. After birth, the fetal zone undergoes atrophy via apoptosis, while the zona glomerulosa and zona fasciculata differentiate from the definitive zone to secrete aldosterone and cortisol respectively, whereas the zona reticularis starts its development postnatally and does not complete development until just before adrenarche (3,4).This process is fully described in the Endotext.org chapter, “Adrenal Cortex: Embryonic Development, Anatomy, Histology and Physiology” (6). The distinctive function of fetal zone and the transition from fetal to adult adrenal function have clinical applications in the very preterm infants who may have transient signs of relative AI after birth (7-9). Furthermore, babies who have a defect in steroidogenesis can present with both adrenal insufficiency and genital atypia (10). The most classic example in this category involves 46,XX babies with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency who present with virilization and adrenal crisis (11). In this chapter, we discuss CAH in the context of AI. Additional information on CAH can be found in the Endotext.org chapter: Congenital Adrenal Hyperplasia (12) https://www.ncbi.nlm.nih.gov/books/NBK278953/. Mineralocorticoid deficiency will be reviewed in the context of primary adrenal insufficiency (PAI), but in-depth coverage of mineralocorticoid deficiency will be included elsewhere as a separate chapter.

PATHOPHYSIOLOGY & CLASSIFICATION

Adrenal insufficiency is classified into primary and central origin (Figure 1) (10,13,14).

Primary AI

Primary AI (PAI) refers to the destruction or dysfunction of the adrenal cortex, often resulting in combined cortisol and aldosterone deficiencies due to injury to both the zona fasciculata and zona glomerulosa (15,16). In cases of destruction, clinical presentation occurs when most of the cortex, >90%, is destroyed. Initial stages may be indolent and may often be followed by an acute stressor leading to clinical presentation and adrenal crisis. In contrast, in cases of dysfunction (i.e. steroidogenesis defects like 21-hydroxylase deficiency causing classical CAH), crisis can occur in the first few weeks of life if the steroidogenic defect is not detected promptly and in the absence of an acute stressor (17). In all cases, decreased glucocorticoid production and decreased negative feedback to the hypothalamus and pituitary result in increased production of ACTH and its pro-hormone pro-opiomelanocortin (POMC) (1). The increase in melanocyte-stimulating hormone (MSH) is responsible for the well-recognized hyperpigmentation present in PAI and often the clinical clue to evolving AI.

Central AI

Central AI involves disorders of the hypothalamic-pituitary region that impair CRH and/or ACTH secretion, and therefore, cortisol production. Aldosterone secretion in the zona glomerulosa of the adrenal cortex is primarily regulated by the renin-angiotensin system rather than ACTH and, thus, remains intact in central AI. Despite the intact mineralocorticoid secretion, hyponatremia may occur due to absence of the glucocorticoid regulated tonic suppression of ADH resulting in volume expansion, a clinical picture similar to the syndrome of inappropriate ADH secretion (SIADH) (18).

SYMPTOMS

Acquired AI can have an insidious onset. Children may present with slowly progressive or nonspecific symptoms, such as anorexia, weight loss, morning nausea or vomiting, and fatigue, which may lead to diagnostic challenges and delayed diagnosis. One cross-sectional, retrospective adult study reported that only 15% of cases were correctly diagnosed at the initial presentation and nearly half had experienced symptoms for more than 1 year before establishing their diagnosis (19). Congenital AI usually presents early in life with signs of adrenal crisis or hypoglycemic seizures. Newborns with central AI may be completely asymptomatic until physical stress elicits an adrenal crisis.

In the pediatric population particularly, specific signs and symptoms should alert the clinician to the possibility of AI. In central AI, central nervous system (CNS) or midline defects may be present. Furthermore, in the neonate, cholestasis may occur due to immature bile acid synthesis and transport (20,21). Virilization and non-palpable gonads associated with the hyperandrogenemia in 46,XX individuals point to classical CAH due to 21-hydroxylase deficiency at birth (22). 46,XY babies with CAH, however, would not present with ambiguity and, thus, may escape medical attention to present with salt-wasting crises between the first and second weeks of life. The inclusion of 17-hydroxyprogesterone measurement in newborn screening programs has enabled detection of these babies prior to the onset of crisis. While the majority of individuals with classical CAH due to 21-hydroxylase deficiency are identified by newborn screening, false negative results and therefore missed diagnosis have been reported (23-26). Moreover, other certain rare enzyme deficiencies are not included in the newborn screen. Thus, a keen clinical index of suspicion remains crucial in cases of CAH who have escaped the newborn screening as experts continue to consider methods to enhance the testing accuracy (27,28). Other associations are discussed in detail under the heading of etiology and are reviewed in Table 1.

In acquired PAI, patients may experience salt craving (i.e., a sign of mineralocorticoid deficiency) and orthostasis and may be noted on examination to have bronze hyperpigmentation, especially in non-sun-exposed areas with prominence in the palmar creases, oral mucosa, skin folds, and areola.

In the case of adrenal crisis, glucocorticoid deficiency may progress to vomiting, muscle weakness, lethargy, hypoglycemia, and ultimately hemodynamic instability. Mineralocorticoid deficiency causes hyponatremia and hyperkalemia and, thus, symptoms may include headache, dizziness, abdominal pain, diarrhea, and ultimately severe dehydration and hypotension. Early detection is critical given the high morbidity and mortality associated with adrenal crisis (29).

ETIOLOGY

Both primary and central AI can arise neonatally due to congenital causes or later in childhood, adolescence and beyond due to acquired causes. In contrast to adults, genetic defects are more likely to be prevalent in infants and children. In recent years, knowledge and understanding of the genetic causes of AI have significantly increased and include every step of the hypothalamic-pituitary-adrenal axis responsible for cortisol and aldosterone synthesis and action (Figure 1 and Table 1).

Figure 1. Genetic defects in various steps along the hypothalamic-pituitary-adrenal (HPA) axis can result in either central or primary adrenal insufficiency depending on the location in the pathway. This figure displays the HPA axis and the location operative in the genetic defect, guiding the assessment of central or primary insufficiency. Genetic causes of central AI can include several genes involved in the development of the pituitary gland (e.g., PROP1, POU1F1 (formerly PIT1), GLI2, HESX1, LHX3, LHX4, SOX3, SOX2, OTX2), and result in multiple combined pituitary deficiencies. Central AI can result from an isolated ACTH deficiency caused by defects in the synthesis of pro-opiomelanocortin (POMC) or its cleavage. Defects of the melanocortin 2 receptor (MC2R) or its accessory protein MRAP result in ACTH-resistance (a.k.a. as Familial Glucocorticoid Deficiency). There are multiple genetic causes leading to primary AI (PAI). They range from genes that are involved in the development of the adrenal gland resulting in adrenal hypoplasia, defects in cortisol synthesis itself, or metabolic and autoimmune diseases (e.g. adrenoleukodystrophy, sphingolipidosis or autoimmune polyglandular syndrome), that result in destruction of the adrenal gland over time. Defects in steroidogenesis involve multiple genes along the pathway of cortisol synthesis and are the most frequent cause of PAI. Finally, PAI can be part of certain syndromes, such as Triple A Syndrome (AAAS) or disorders associated with oxidative stress.

Causes of Primary AI

CONGENITAL CAUSES

Disorders of Steroidogenesis

Disorders of steroidogenesis include 1) defects in the cholesterol biochemistry including Smith-Lemli-Opitz (DHCR7) (30), 2) early steroidogenesis defects such as congenital lipoid adrenal hyperplasia (STAR) and CYP11A1 mutations (31,32), and 3) defects within the adrenal gland causing CAH, the most common being CYP21A2 (21-hydroxylase deficiency) and rare forms including CYP11B1 (11β-hydroxylase deficiency), 3HSD2 (3β-hydroxylase deficiency), CYP17A1 (17α-hydroxylase deficiency), and POR (Cytochrome P450 oxidoreductase deficiency) (12) (See Fig 2 for depiction of adrenal steroidogenesis, Table 1 for further information on clinical presentations and associations and refer to the Endotext chapter on CAH for further details.)

Figure 2. The classical and backdoor pathways of adrenal steroidogenesis: The classical pathway is highlighted in blue, and the backdoor pathway is highlighted in orange. In the classical pathway, five enzymatic steps are necessary for cortisol production. In the first step of adrenal steroidogenesis, cholesterol enters mitochondria via a carrier protein called steroidogenic acute regulatory protein (StAR). ACTH stimulates cholesterol cleavage, the first and rate limiting step of adrenal steroidogenesis. The five enzymes required for cortisol production are cholesterol side chain cleavage enzyme (SCC), 17α-hydroxylase, 3β-hydroxysteroid dehydrogenase (3βHSD2), 21-hydroxylase, and 11β-hydroxylase. The backdoor pathway is an alternative pathway producing dihydrotestosterone. The enzymes include 5α-reductase 1, aldo keto reductases, retinol dehydrogenase RoDH, 17β-hydroxysteroid dehydrogenases, 17α-hydroxylase. (Figure from CAH Endotext Chapter).

Adrenal Hypoplasia

Underdevelopment of the adrenal glands may occur as an X-linked genetic disorder (DAX1/NROB1 – X-linked AHC) and results in both glucocorticoid and mineralocorticoid deficiency (33,34). X-linked AHC is associated with hypogonadism and in some cases, muscular dystrophy. Adrenal hypoplasia can be seen as part of syndromic undergrowth disorders including IMAGe and MIRAGE (35), or in association with gonadal dysgenesis and sex reversal such as NR5A1 (formerly SF1) (34) and SeRKAL syndrome (WNT4) (36) (see Table 1).

ACTH Resistance-Like Conditions

ACTH-resistance, known as Familial Glucocorticoid Deficiency (FGD), is another childhood cause of PAI. It is caused by defects of the melanocortin 2 receptor (MC2R) or its accessory protein at the adrenal gland rendering it unresponsive to ACTH action (Figure 1 and Table 1). Given the specific operative nature of ACTH resistance, concerns for mineralocorticoid insufficiency are rare (37). In FGD type 1, the MC2R receptor is dysfunctional. Patients may present with hyperpigmentation, jaundice, hypoglycemia, and early adrenal crisis or later in life with hyperpigmentation and fatigue (38). In FGD type 2, the MC2R receptor is absent. These cases are due to deficiency of the MRAP (melanocortin 2 receptor accessory protein) which assists in trafficking of the MC2R receptor to the cell surface.

Other disorders associated with ACTH resistance include those associated with oxidative stress (most commonly defects in nicotinamide nucleotide transhydrogenase [NNT] but also described in thioredoxin reductase 2 [TNXRD2]), Triple A Syndrome (Allgrove syndrome due to disruption of the Aladin protein [AAAS] ACTH resistance/Addison disease, alacrima (absence or deficiency of ocular tears), and achalasia of the esophagus)(37,38), and disruption of mini-chromosome maintenance 4 (MCM4)(37-39).

Metabolic Conditions

Metabolic conditions associated with PAI include mitochondrial diseases – large gene deletions such as Kearns-Sayre and Pearson as well as single gene disorders (MK-TK, MRPS7, QRSL1, NDUFAF5, GFER), lysosomal storage disorder (Wolman), sphingolipidosis (SGPL1 deficiency), and adrenoleukodystrophy(40-42). Descriptions and associations can be found in Table 1. In adrenoleukodystrophy, neurologic features can prompt identification. However, neurologic manifestations are absent in adrenal only forms of the disease. Adrenoleukodystrophy is included in the newborn screening programs in some states in the US. However, screening is not yet universal in the US and is rare in other countries. Consequently, a high index of suspicion should be maintained for all boys with unknown cause of PAI, and very long chain fatty acids (VLCFA) should be measured. Prompt diagnosis aids in therapeutic decision- making including hematopoietic stem cell transplant as well as more recent approval for gene therapy (43,44).

Zellweger Spectrum disorders consist of a group of peroxisomal related gene disorders that result in neurological dysfunction, hepatic dysfunction, renal cysts and PAI. Screening for PAI is recommended after the first year of life(45).

AQUIRED CAUSES

Autoimmune Conditions

Addison’s disease (autoimmune adrenal insufficiency) is the most common cause of AI in adolescents and adults with a median age of presentation at 11 years (15). While less common in young children, it can be seen in association with other endocrine dysfunctions (poly-endocrinopathy). The best described monogenic cause is related to a defect in the AIRE gene and results in Autoimmune Polyglandular Syndrome type 1 (APS1). Presenting features often include mucocutaneous candidiasis and hypocalcemia related to hypoparathyroidism. In contrast, APS2 is typically of polygenic inheritance and is associated with autoimmune thyroiditis and diabetes mellitus with abnormalities in the DQ and DR genes. Adrenal insufficiency related to APS2 has a later age of onset of about 35 years of age, although pediatric cases do occur (15).

Other Causes of Primary AI

Other acquired causes include hemorrhagic, infectious, and infiltrative conditions. Adrenal hemorrhage should only result in adrenal insufficiency if both glands are affected extensively, as 10% of remaining function is sufficient to retain adrenal activity (46). Unilateral hemorrhage should not cause adrenal insufficiency as the single remaining gland is able to compensate. While adrenal hemorrhage occurs fairly often in traumatic birth delivery, few require treatment and of those that do, most resolve within 3-9 months (47). Waterhouse-Friderichsen syndrome is bilateral adrenal hemorrhage of infectious etiology and described with N. meningitides, streptococcus pneumoniae, and other bacterial infections (48). This adrenal insufficiency may also be reversible upon treatment of the primary infection supplemented with cortisol as needed. In contrast, tuberculous adrenal disease results in necrosis of the gland and is often irreversible unless discovered very early in the course of tuberculosis (49).

Several drugs increase cortisol clearance and may precipitate adrenal crisis or require a dose increase in patients with AI on daily glucocorticoid replacement. These include CYP3A4 inducers such as rifampin, mitotane, carbamazepine, and St. John’s wort. Initiation of growth hormone and T4 replacement may also increase cortisol metabolism (50). Medications used for treating excessive glucocorticoid secretion (Cushing’s disease) that directly block steroidogenesis (ketoconazole, etomidate, metyrapone, osilodrostat) and the glucocorticoid receptor (mifepristone) can induce adrenal insufficiency (51).

Central AI

CONGENITAL CAUSES

Combined Pituitary Hormone Deficiency

Many genetic causes of hypopituitarism (consisting of loss of ACTH with other anterior pituitary hormones such as GH, TSH, LH/FSH) have been elucidated including PROP1, POU1F1 (formerly PIT1), GH1, GLI2, HESX1, LHX3, LHX4, SOX3, SOX2, OTX2 and others (20,21,52). These can result in hypopituitarism on its own (non-syndromic) or in a constellation of other associated phenotypic features (syndromic), commonly involving midline defects. Despite a genetic etiology, some conditions including PROP1 and GH1 may manifest with ACTH insufficiency later in life. As such, close monitoring of the HPA axis in the setting of other pituitary deficiency is crucial (53).

Isolated ACTH Deficiency

Isolated ACTH deficiency can occur due to disruption of TBX-19 (formerly known as TPIT, required for pro-opiomelanocortin protein transcription), due to defects in pro-opiomelanocortin (POMC) or pro-hormone convertase-1 (PC-1/PCSK1) (20). The classic findings of red hair and pale skin and hyperphagic obesity in POMC mutations are related to the inability to produce pigmentation and regulate hunger in the absence of α melanocyte stimulating hormones. Another syndromic description involving ACTH deficiency includes DAVID (deficit in anterior pituitary function and variable immunodeficiency) syndrome which occurs in 2/3 of NFKB2 mutations.

AQUIRED CAUSES

Brain Lesion or Injury, Infiltrative Disease

Traumatic brain injury, brain tumor, infiltrative (histiocytosis, iron overload), and inflammatory (hypophysitis) disorders can result in pituitary dysfunction and impaired ACTH secretion; additional pituitary hormone deficiencies can also occur frequently with these conditions. Diagnosis of AI in these settings should therefore prompt a complete pituitary workup, including evaluation for central hypothyroidism, growth hormone deficiency, and hypogonadotropic hypogonadism (if in a pubertal child). Pituitary surgery may result in central AI post-operatively and during recovery. In contrast, in cases of brain radiation or developmental differences of the pituitary gland, AI may occur over time, and longitudinal assessment is suggested for affected individuals. Traumatic brain injury has been associated with central adrenal insufficiency in up to 14% of individuals with moderate to severe TBI and 6% of athletes with history of concussion with data coming from the adult population (54,55). Data in children have shown central AI in up to 47% of individuals at 3 months and recovery by 12 months after injury (56).

Transient (Hypothalamic Suppression)

Glucocorticoid induced - Oral glucocorticoid administration above physiologic doses (i.e. Hydrocortisone 8-10mg/m²/day or equivalent) is associated with HPA axis suppression and subsequent adrenal atrophy after 2-4 weeks of daily use (57,58). Although there is significant interindividual variability, risk factors for AI include dose, frequency, and potency of the used glucocorticoid (59). Adrenal suppression can also be seen with inhaled or intranasal glucocorticoids, especially if their use is combined with intermittent use of oral formulations. Screening for AI has been suggested for children taking high doses of inhaled glucocorticoids (>500µg/day of Fluticasone or equivalent) for more than 6 months (60). Particular attention should be paid to the combination of glucocorticoid therapy and CYP3A4 inhibitors, such as grapefruit juice, as the latter can reduce glucocorticoid clearance, and therefore, augment their efficacy.

Adrenal function recovers once glucocorticoids are discontinued. Time to recovery is variable from 1 to many months, and can be affected by length of exposure, dose, frequency, and glucocorticoid potency (61-63). As such, recovery should be monitored, and stress dosing instructions provided until adrenal recovery.

Through a similar mechanism, successful treatment of Cushing’s syndrome and Cushing’s disease by resection of the pituitary or adrenal lesion results in a temporary adrenal insufficiency, necessitating replacement until recovery is demonstrated.

Other medications - Various medications have been shown to suppress the HPA axis. This is more frequently seen with long term opiate use (Opiate induced adrenal insufficiency - OIAI) due to tonic inhibition of the HPA axis – in which studies have shown 9 to 29% of individuals with adrenal suppression, potentially with high risk associated with younger age (64). There have also been cases of adrenal suppression with short term opiate use (65). Other medications responsible for adrenal suppression include somatostatin analogues, antipsychotics, and antidepressants.

Critical illness- such as sepsis or severe trauma, elicit a “fight-or-flight” or “stress” response that involves multiple physiological processes, including release of catecholamines and activation of the HPA axis. HPA axis activation results in a rapid rise in circulating ACTH and cortisol. The concept of “relative” AI during critical illness was introduced in the early 2000’s based on adult data of patients with septic shock who demonstrated an inadequate cortisol response to endocrine testing. The term “relative AI” was later replaced by “critical illness related corticosteroid insufficiency (CIRCI)” as these patients typically have “inappropriately” low ACTH and cortisol levels in response to stress.

Changes in adrenal function during critical illness are not fully understood (66). However, current research indicates that there is an initial brief HPA axis activation in response to a critical illness followed by a series of adaptive events that include a reduction in cortisol-binding globulin (CBG)/albumin leading to an increase in free cortisol, prolonged cortisol half-life due to suppressed metabolism in liver and kidneys, and tissue-specific changes in glucocorticoid receptor action (66). These peripheral adjustments increase systemic cortisol availability, and with prolonged illness, may result in central HPA suppression. From the clinical standpoint, endocrine testing (i.e. measurement of serum cortisol at baseline or after ACTH stimulation) can be challenging due to reductions in CBG, with one study noting this reduction lasting 7-8 days in adult patients (67). In terms of treatment, randomized controlled trials of stress dose hydrocortisone in critically ill adults showed inconsistent results on long term mortality (68). Hydrocortisone administration was found to have a positive effect on blood pressure, which can be related the pharmacologic effects of high dose glucocorticoids rather than treatment of CIRCI (66).

Pediatric data on CIRCI are limited. Current pediatric guidelines do not recommend hydrocortisone treatment for children with sepsis who are hemodynamically stable after fluid resuscitation but can be considered in those with fluid-refractory, inotrope-resistant shock (69).

Relative AI of the newborn - Very preterm infants may experience refractory hypotension that is unresponsive to fluid resuscitation and inotropic support but responds to treatment with glucocorticoids (7-9). No other apparent cause, like sepsis, is identified in these cases and electrolyte abnormalities indicative of a mineralocorticoid defect can be observed. It has been suggested that these infants have an attenuated cortical response to stress or relative AI. The term transient adrenocortical insufficiency of prematurity (TAP) has also been used as this phenomenon resolves within the first couple weeks of life (8). Immaturity of the HPA axis both at the level of hypothalamus, pituitary, and the adrenal gland itself have been implicated as the underlying pathophysiology. A late-onset GC -responsive circulatory collapse that occurs within the first 2 weeks of life and responds to therapy with glucocorticoids has also been described (70).

Formal diagnosis is challenging given studies failing to show association of low cortisol concentrations with adverse outcomes (71) while others identifying an association between high cortisol values with both morbidity (intracranial hemorrhage and cerebral palsy) and mortality (72,73).

Neither baseline nor stimulated values of cortisol have shown diagnostic benefit.

Cortisol secretion markedly increases during parturition to assist with lung maturation and transition to life after birth. There are data to suggest that very preterm infants who develop bronchopulmonary dysplasia (BPD) often have relative AI after birth. To address this concern, early low-dose hydrocortisone therapy as prophylaxis for AI was found to be beneficial for survival without BPD, although the treatment was associated with increased risk for spontaneous gastrointestinal perforation (74).

TABLE 1. CAUSES OF ADRENAL INSUFFICIENCY IN CHILDREN
Congenital - Central	Combined pituitary deficiencies	Non syndromic (PROP1, POU1F1)	Variable presentations associated with single or multiple pituitary defects including ACTH deficiency. May include hypoglycemia and/or microphallus
	Combined pituitary deficiencies	Syndromic	· Associated syndromes include optic cell hypoplasia / septo-optic dysplasia, microphthalmia (HESX1, SOX2, OTX2) and various CNS malformations (i.e., holoprosencephaly) with midline defects. · Syndromes with hypothalamic dysfunction (e.g., ROHHAD syndrome, Prader- Willi syndrome) can rarely be associated with ACTH deficiency.
	Isolated ACTH deficiency	Defects in TBX19, POMC or PCSK1	· Can present in the newborn with hypoglycemic seizures and jaundice. · Associated features (POMC): red hair, hyperphagia/obesity. MC4R agonists can be leveraged to treat obesity. · Associated features (PCSK1): malabsorptive diarrhea, obesity, and hypogonadism
Acquired - Central	Brain lesion or injury	Examples: Tumor, hemorrhage, irradiation	· Usually associated with additional pituitary defects · Immune checkpoint inhibitors can be associated with hyper-autoimmunity and cause hypophysitis and hypopituitarism.
	Infiltrative disease	Examples: iron overload (due to transfusions in thalassemias, hemochromatosis), sarcoidosis, Langerhans cell histiocytosis	· Usually associated with additional pituitary defects. · Symptoms specific to the underlying causative disorder.
	Transient (hypothalamic suppression)	Glucocorticoid therapy	· The most frequent cause of adrenal insufficiency. · Adrenal function recovers with discontinuation of daily glucocorticoids.
		Medications	· Opiates, somatostatin analogues, antipsychotics and antidepressants
		Treatment of Cushing	· Permanent: hypophysectomy · Transient: post operative for unilateral adrenalectomy, or pituitary lesion
		Critical illness -related corticosteroid insufficiency (CIRCI)	Characterized by a series of adaptions of the HPA axis in response to critical illness, which are still not clearly understood. The beneficial effects of glucocorticoids in sepsis are likely related their anti-inflammatory and blood -pressure support effects rather than treatment of adrenal insufficiency.
Congenital - primary	ACTH resistance	Familial Glucocorticoid Deficiency	Type 1 (MC2R): Presents first weeks of life with severe hypoglycemia, prolonged jaundice
		Familial Glucocorticoid Deficiency	Type 2 (MRAP): Presents first few months of life with AI
		Syndromic	Triple A (Allgrove) syndrome (AAAS): Associated with alacrimand achalasia of the esophagus presenting in childhood or 2^nd decade of life
		Syndromic	Disorders associated with oxidative stress (NNT, TNXRD2)
	Congenital Adrenal Hypoplasia	X- linked Adrenal Hypoplasia (DAX-1/NR0B1)	· Can present with salt losing early on or may present later in childhood · Additional features: hypogonadotropic hypogonadism. · Associated with a larger Xp21 contiguous gene deletion that can result in Duchene Muscular Dystrophy and Glycerol Kinase deficiency · Growth hormone insufficiency in a small subset
		Syndromic	IMAGe syndrome (gain of function CDKN1C, POLE1): Additional features: IUGR, metaphyseal dysplasia, GU anomalies
			MIRAGE syndrome (gain of function SAMD9): Additional features: Myelodysplasia, infections, restriction of growth, GU variations, enteropathy
			NR5A1 mutation and SeRKAL syndrome (WNT4): Associated with gonadal dysgenesis and sex reversal in 46XX individuals
			Rare reports: Pena-Shokeir syndrome type 1 (DOK7, RAPSN), pseudotrisomy 13, Galloway-Mowat (WDR73), Pallister Hall (GLI3) and Meckel-Gruber(MKS1)
	Disorders of steroidogenesis	Congenital Adrenal Hyperplasia (CAH)	Due to defects in 21-hydroxylase (CYP21A2): · The most common cause of PAI in neonates. · Presents with salt wasting adrenal during 1^st month of life crisis and genital virilization in 46XX individuals. · Biomarker: elevated 17-hydroxyprogesterone levels. Included in the state newborn screening in US
		Congenital Adrenal Hyperplasia (CAH)	Due to defects in 11β-hydroxylase (CYP11B1), 3β-hydroxysteroid dehydrogenase type 2 (3HSD2), 17α-hydroxylase/17,20-lyase (CYP17A1), P450 oxidoreductase (POR): · Variable phenotype depending on the defect. Salt wasting and genital atypia can be present. · Biomarker: steroid precursor that accumulates above the specific enzymatic defect
		Defects in cholesterol biochemistry	· Smith-Lemli-Opitz (DHCR7). Additional features: Microcephaly, cleft palate, syndactyly, polydactyly, congenital heart, atypical genitalia with undescended testis. · Biomarker: elevated 7-dehydrocholesterol. AI is rare · Wolman disease (LIPA). Additional features: lysosomal storage disorder, hepatosplenomegaly, adrenal calcifications, failure to thrive
		Early steroidogenic defect	Congenital lipoid adrenal hyperplasia (STAR), P450 Side Chain Cleavage (CYP11A1) mutations: · Associated with salt wasting and under-virilization of 46XY individuals. · Operative in adrenal and gonadal steroidogenesis Salt wasting crisis in severe P450scc deficiency presents at 7-10 days while in STAR deficiency, the onset is more insidious, after 3-4 weeks of age Partial enzyme defect may have less severe presentation
	Metabolic disorders	Sphingosine-1-Phosphate Lyase (SGPL1) deficiency	· Results from impaired breakdown of sphingosine 1-phosphate. · Additional features: adrenal calcifications, nephrotic syndrome. Ichthyosis, neurologic dysfunction.
		Adrenoleukodystrophy	· X- linked (ABCD1). Usually presents in childhood. Associated with progressive neurologic deterioration. · Zellweger Spectrum disorders (PEX). Associated with neonatal adrenoleukodystrophy, hepatomegaly, chondrodysplasia punctuate, hypotonia, seizures. · Biomarker: Elevated Very Long Chain Fatty Acids.
		Wolman	· LIPA mutation – lysosomal storage disorder with foamy lipid droplet accumulation, adrenal calcification and malabsorption
		Mitochondrial Disorders	Variable Presenting features.
Acquired – Primary	Autoimmunity	Addison disease	· The most common cause of acquired PAI. Presents in childhood. · Biomarker: 21-hydroxylase antibodies · Can be part of autoimmune polyglandular syndrome (APS) type I (AIRE) or type II (polygenic) o APS 1: other associations include: hypoparathyroidism, chronic mucocutaneous candidiasis, ectodermal dystrophy, autoimmune hepatitis, hypogonadism, pernicious anemia. o APS2: other associations include autoimmune thyroiditis, diabetes mellitus
	Adrenal damage	Hemorrhage, Infectious, Infiltration (Tuberculosis, HIV, CMV)	Infectious: Tuberculosis, HIV, CMV Infiltration: neuroblastoma
	Transient	Relative adrenal insufficiency of the newborn	Described primarily in preterm sick infants within 2 weeks of life with signs of refractory hypotension in the absence of apparent cause and associated with electrolyte abnormalities (hyponatremia, hyperkalemia). May be related to immature adrenals in the very preterm infants.
		Treatment of Cushing	· Permanent: Bilateral adrenalectomy · Medication induced: ketoconazole, metyrapone, osilodrostat, etomidate, mifepristone
		Medication induced	CYP3A4 inducers: rifampin, mitotane, carbamazepine and St. John’s wort

Mimickers of Adrenal Insufficiency

In the introduction to this chapter, we discussed the risk of delayed diagnosis of AI given the potential overlapping symptoms of glucocorticoid deficiency with other illnesses. In the same vein, other critical illnesses including sepsis and cardiovascular disease may be mistaken for adrenal crisis, especially in the setting of hydrocortisone responsiveness. In this case, hydrocortisone’s inotropic properties may lead to symptomatic improvement but may not be diagnostic of underlying AI.

Further, salt wasting adrenal crisis has findings overlapping with other salt-losing crises in infants including those due to kidney inflammation, infection, and obstruction – presenting with vomiting, hyponatremia, and hyperkalemia due to aldosterone resistance (a transient pseudo hypoaldosteronism) (75,76).

EPIDEMIOLOGY

Primary Adrenal Insufficiency (PAI)

PAI is a rare disease. Its incidence in children is not well established. An epidemiologic study from Finland observed a cumulative incidence of 10/100,000 at 15 years and 13/100,000 at 20 years (15). Studies from Europe that include both children and adults describe a similar prevalence (71).

In adults, the most common cause of PAI is autoimmune disease. In contrast, genetic defects are more prevalent in children. CAH due to 21-hydroxylase deficiency occurs in about 1/15,000 births and is the most frequent cause of PAI in children accounting for 50 to 86% of cases (15,77). As such, the age-related incidence of PAI in childhood is higher in the first year of life and decreases afterwards (15). Autoimmune disease is reported as the second most common cause, either as an isolated disease or as a manifestation of a poly endocrinopathy (i.e. APS 1 or 2) (77,78). Whereas most CAH cases are identified in the newborn period, autoimmune disease typically presents after the first couple years of life. X-linked adrenoleukodystrophy (XALD) is another cause of PAI during childhood with a prevalence in newborn males of about 1/20,000, approximately 80% of whom will develop PAI (79).

In recent years, the number of recognized genetic causes of PAI have significantly increased(Figure 1) (13). Genetic testing for PAI has a high diagnosis rate as illustrated by recent studies of children with PAI of unknown etiology, where genetic analysis established the diagnosis in most of the patients (80,81). The most frequent genes involved in these series were MC2R, NR0B/DAX and CYP11A1.

Central AI

Central AI is also quite rare with most epidemiologic data available in adult populations. The reported occurrence is about 14-28/100,000 individuals, with greater than 50% occurring because of a pituitary tumor (82-84). A Finnish epidemiologic study reporting single center data over a period of 30 years is one of the only studies of incidence in pediatrics. The study looked specifically at combined pituitary hormone deficiencies, without clearly distinguishing AI, and observed similar proportions in pediatric in comparison with previous adult studies, with 61% of hypopituitarism being acquired and the other 39% congenital (85). Craniopharyngiomas comprise the bulk of pediatric pituitary tumors, and similarly account for one quarter of acquired hypopituitarism, with gliomas following at just over 10% (85). Up to 30% of patients with craniopharyngiomas will present with AI due to the lesion itself prior to surgical manipulation while 77-90% will have post-surgical AI (86,87).

Glucocorticoid induced AI is the most common cause of AI in both children and adults. Glucocorticoids are extensively used in clinical practice for treatment of various disorders, such as asthma, autoimmune and inflammatory diseases, and cancer (88). In US, approximately 1% of the adult population is on oral glucocorticoids and rates increase in the elderly population (89), while similar rates in children are not well established. In adults, AI has been observed around 48% with oral administration and 7.8% with inhalation (57). Rates of AI in children on inhaled glucocorticoids have been reported around 10%, but results may vary depending on the study (90,91). Adrenal crisis and related deaths have been described in children treated with inhaled glucocorticoids(92,93).

DIAGNOSTIC APPROACH TO A PATIENT WITH SUSPECTED AI

Our diagnostic and differential approach to a child with suspected AI is summarized in Figure 2. Clinical signs and symptoms of AI are non-specific and require a high index of suspicion; when present, the suspected diagnosis needs to be confirmed with appropriate laboratory evaluation. Evaluation typically starts with measurement of serum ACTH, cortisol, and electrolytes. In the case of an emergency, such as suspected adrenal crisis, laboratory evaluation can only be interpreted if obtained prior to administration of glucocorticoids as therapy can influence test results.

The goals of the initial laboratory assessment are to confirm the diagnosis of AI, and then, understand if AI is primary or central. Most cases of PAI, but not all, involve combined glucocorticoid and mineralocorticoid deficiencies. In the case of PAI, therefore, mineralocorticoid function must be assessed with measurements of serum electrolytes, aldosterone, and renin (Figure 2). Details on laboratory assessment and additional dynamic testing are included in the LABORATORY EVALUATION section of this chapter.

The next step is to pinpoint the specific etiology so that management can be individualized. Clinical presentation and symptoms can suggest an underlying etiology and guide additional laboratory assessment. For example, salt wasting adrenal crisis in a newborn should prompt measurement of serum 17-hydroxyprogesterone concentrations to rule out CAH. Vitiligo in a child with PAI suggests an autoimmune process, and the underlying etiology can be confirmed with measurement of 21-hydroxylase antibody titers. AI in a boy with neurological manifestations points to adrenoleukodystrophy and calls for measurements of VLCFA. In many instances, however, the specific etiology cannot be identified. In such cases, genetic testing may be very helpful.

Figure 3. A proposed approach in the diagnosis and differential of AI in children
GC: glucocorticoid. MR: mineralocorticoid. Na: sodium. K: potassium.

LABORATORY EVALUATION

Assessment of Glucocorticoid Function

BASELINE ASSESSMENT OF CORTISOL AND ACTH CONCENTRATIONS

The diagnosis of PAI can be made by measurement of morning ACTH and cortisol concentrations and by demonstrating an inappropriate high ACTH concentration for the cortisol value (Figure 2). Typically, a cortisol value of <5mcg/dL (140nmol/L) associated with an ACTH level > x2 above upper normal range indicates PAI (16). A low morning cortisol value associated with an inappropriately normal ACTH concentration may indicate central AI (94). It is suggested that an 8am cortisol values of <3 mcg/dL (82.8 nmol/ L) in the absence of an elevated ACTH concentration is indicative of central AI (94). Establishing the diagnosis of central AI can be challenging because ACTH concentrations are frequently in the low normal/normal range. The presence of additional pituitary defects increases the risk for AI and needs to be considered when evaluating a child with possible central AI.

Assessment of adrenal function, such as in individuals at risk for glucocorticoid-induced AI, can also start with a measurement of morning cortisol (58,95). Adult studies suggest that an 8am cortisol value of >12mcg/dL can predict normal adrenal function (96,97). It is recognized, however, that various guideline and practice recommendations point to slightly different cut-offs (58,94). Further, the suggested cut-offs need to be interpreted by taking into account the cortisol assay that is used. Of note, the listed cut-offs also do not apply for patients on oral contraceptives and/or estrogen replacement as these medications increase CBG concentrations, and therefore, cortisol values (98).

Morning cortisol values may be unreliable in the newborn as the diurnal pattern of cortisol secretion may take few months to become established (99). Baseline cortisol values have been extensively studied in the sick preterm infant and were not found to be predictive of AI or adverse outcomes (71-73). Furthermore, high cortisol values during first week of life were associated with intracranial hemorrhage and cerebral palsy later in life (73).

DYNAMIC OR STIMULATION TESTS

Stimulations tests are typically performed when the diagnosis of AI needs confirmation. The ACTH (a.k.a. corticotropin or Cosyntropin or Synacthen) stimulation test is the primary test performed in children. Historically, the insulin tolerance test (ITT) has been considered the gold standard test for the diagnosis of AI, but its use has been abandoned as it is associated with severe hypoglycemia (1,16,95). The overnight metyrapone test, an alternative to ITT, is also not routinely performed in children because of potential significant adverse effects (16,95). The CRH (corticotropin releasing hormone) stimulation test has been used in the diagnosis of central AI but there is no evidence that it has greater diagnostic accuracy than ACTH testing in pediatrics and CRH is no longer commercially available (100).

The ACTH test involves the intramuscular or intravenous administration of cosyntropin, a synthetic fragment of ACTH with full biologic activity, along with cortisol measurements at baseline and after cosyntropin administration. Although differences in practice protocols have been described, a common protocol entails cosyntropin doses of 15mcg/kg in infants, 125mcg in children <2years and 250mcg in children older than 2 years (16). Cortisol concentrations are usually measured at 30- and 60-minutes post stimulation, although again, practice variations have been described with the use of the “short” ACTH test that includes cortisol measurement at baseline and only at 30 min after stimulation(101).

The standard dose (250mcg) ACTH test has been validated against the ITT; it has good diagnostic accuracy for PAI but only moderate for central AI (102,103). The low dose ACTH test that involves administration of 1mcg of cosyntropin was developed as an alternative that may offer greater sensitivity in the diagnosis of central AI. Like adult data, however, meta-analysis of pediatric studies indicates that both standard- and low- dose ACTH tests have similar diagnostic accuracy with high specificity but only moderate sensitivity for the diagnosis of central AI (102). These findings suggest that the clinical picture needs to be considered when interpreting ACTH test results. Furthermore, the low dose ACTH test bears technical challenges related to potentially erroneously low administered dose because it requires dilution and cosyntropin is known to adhere to plastic tubing used in intravenous tests (103).

Regardless of the standard- or low- dose ACTH test used, the diagnosis of AI is based on peak cortisol concentrations of 18mcg/dL at either 30 or 60 minutes after cosyntropin (16). The cortisol cut-off of 18mcg/dL was established using cortisol assays of previous methodology that are no longer in use, and therefore, is not applicable in today’s clinical practice. Cortisol measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) is currently considered the gold standard, yet cortisol immunoassays remain widely used in most facilities. Although there no official guidelines or consensus statements at the moment, adult data support revised cortisol cut-offs at 30 min post cosyntropin of around 15mcg/dL using LC-MS/MS and second-generation immunoassays such as Elescys II (Roche Diagnostics) and Access (Beckman Coulter) (101,104,105) . Collectively, the adult data call for revised cortisol cut-offs specific to the assay that is used to avoid overdiagnosis of AI. Limited pediatric data show similar findings as in adults (106).

Literature deriving primarily from critical care medicine uses a cortisol rise of >9mcg/dL during ACTH stimulation to define AI. This criterion is greatly influenced by the baseline cortisol value and has been abandoned (66,95). The ACTH test may miss the diagnosis of AI in newborns with a central defect in ACTH secretion (e.g., septo-optic dysplasia or congenital panhypopituitarism) if it is performed within 1-2 weeks after birth. These babies have normal fetal adrenal function driven by placental CRH stimulation, and therefore, normal response to ACTH stimulation shortly after birth(107). However, they develop adrenal atrophy and insufficiency within the first 2 months of life. Similarly, the ACTH test can be falsely negative if the testing is done during the early stages of AI due to exogenous glucocorticoids or a pituitary/CNS insult and before the atrophy of the adrenals has occurred (95). A high index of suspicion and repeat testing may be needed in such cases. The ACTH test results can be significantly influenced by CBG concentrations. Estrogens are well known to increase CBG concentrations, and estrogen containing oral contraceptives can increase the cortisol cut-off after ACTH stimulation by approximately 10mcg/dL (98,108). Conversely, disease states such as acute illness, cirrhosis, and nephrotic syndrome lead to decreased CBG concentrations and potential overdiagnosis of AI (98). Rare genetic syndromes, such as congenital CBG deficiency or familial glucocorticoid resistance, can also affect baseline as well as stimulated cortisol measurements.

The standard-dose ACTH test is also performed when a disorder of steroid biosynthesis, such as CAH, is suspected. In addition to cortisol, adrenal steroids are measured at baseline and 60-minute post stimulation. Specific nomograms based on 17-hydroxyprogesterone values have been developed for the diagnosis of CAH due to 21-hydroxylase deficiency (12).

Assessment of Mineralocorticoid Function

Electrolytes abnormalities that include hyponatremia in combination with hyperkalemia and acidosis should raise suspicion for a mineralocorticoid defect. The simultaneous measurement of plasma aldosterone and renin is important for the diagnosis of a mineralocorticoid defect. An inappropriate low aldosterone concentration in the presence elevated renin levels indicates mineralocorticoid deficiency. In such instances, the treating physician needs to carefully assess the glucocorticoid function of the child to determine whether the child has combined deficiencies, such as seen in CAH, or an isolated mineralocorticoid defect, such as seen with aldosterone synthase deficiency. Conversely, markedly elevated aldosterone and renin levels in a child with hyponatremia and hyperkalemia suggest mineralocorticoid resistance, such as pseudo-hypoaldosteronism(109).

Aldosterone and renin concentrations are higher in the newborn and can be affected by prematurity (110-112). Newborns at term have a state of partial aldosterone resistance and hence concurrent high aldosterone values in their blood, that resolve in the first few months of life, whereas babies with severe prematurity have an initial defect in aldosterone secretion followed later by the appearance of physiologic aldosterone resistance (110-112). These physiologic changes need to be considered when assessing mineralocorticoid function in the first few months of life. Lastly, some children with PAI, like those with adrenoleukodystrophy, develop glucocorticoid deficiency initially, while they retain mineralocorticoid activity (79); ongoing monitoring with measurements of serum electrolytes, plasma aldosterone and renin concentrations are required to determine the need for starting mineralocorticoid replacement.

Supporting/Miscellaneous Testing

Once AI is documented, additional testing based on patients’ history and symptomatology can pinpoint the specific cause. For example, presence of genital atypia suggests a disorder of steroidogenesis, and elevated concentrations of specific steroid precursors can determine the enzymatic defect (e.g. 17-hydroxyprogesterone in 21-hydroxylase deficiency or 11-deoxycorticosterone in 11β-hydroxylase deficiency) (12). Elevated VLCFA can establish the diagnosis of adrenal leukodystrophy in a boy with AI and neurological manifestations. Positive antibody titers for 21-hydroxylase indicates autoimmune Addison disease. Nonetheless, the underlying cause can be frequently tentative. To this end, genetic testing has become increasingly important. Testing can be tailored according to clinical suspicion and varies from single candidate gene analysis to targeted “panels” or whole exome sequencing (WES). In general, genetic testing for AI has a high diagnostic success rate and can be very helpful in complex cases where diagnosis remains otherwise uncertain (80).

TREATMENT

Hormone Replacement

The goals of daily replacement therapy are to avoid symptoms of adrenal insufficiency while securing optimal growth and weight gain, and appropriate puberal progression.

GLUCOCORTICOID REPLACEMENT

The glucocorticoid of choice for children is hydrocortisone, typically given three time daily (16). The physiologic daily cortisol secretion is approximately 5-8mg/m²/day (113). In practice, typical replacement doses are 8-10mg/m²/day, except in CAH which frequently requires supraphysiologic doses to suppress adrenal androgen secretion. The dose distribution during the day usually mimics the diurnal pattern of cortisol secretion with the largest dose given upon awakening, the second dose around noon and the last dose at early evening to avoid overnight hypercortisolemia, which may lead to sleep disturbances, stunted growth, or insulin resistance. A reverse circadian hydrocortisone administration has been used in CAH to suppress the overnight rise in ACTH. This regimen has been criticized as non-physiologic. Individuals with central AI can also be treated with a twice a day regimen (94), with the first larger dose given upon awakening and the second smaller dose in late afternoon. It is our practice to place children with central AI on a twice a day glucocorticoid replacement regimen, acknowledging that this procedure is based on adult recommendations, while pertinent pediatric data or guidelines are lacking. Specific to children with glucocorticoid-induced AI, hydrocortisone replacement twice a day with the second dose administered in late afternoon can be beneficial and facilitate recovery of the adrenal axis as it avoids nighttime hypercortisolemia and suppression of ACTH secretion.

Challenges with daily hydrocortisone replacement include its short half-life of 60-90min and significant variability in clearance among patients, which result in alternating periods of hypo- and hyper-cortisolemia while on treatment. A modified release hydrocortisone (Plenadren by Takeda Pharmaceuticals International AG Ireland) was designed as a more physiologic and convenient alternative (114). Plenadren provides hydrocortisone released in two phases (i.e., an initial phase of immediate release followed by a phase of extended release) and is given as a once-a day dose upon awakening. It is available in Europe but not in the USA. An additional concern with hydrocortisone administration in young children involves difficulties in dose titration as hydrocortisone is not available in tablets less than 5 mg. To overcome this barrier, hydrocortisone microgranules that provide doses as low as 0.5 mg have been introduced in the market (115). Hydrocortisone microgranules have 1:1 bio equivalency with the standard immediate release hydrocortisone. Finally, it should be kept in mind that certain drugs, like carbamazepine or phenytoin, can induce CYP3A4 in the liver and increase hepatic hydrocortisone clearance requiring higher hydrocortisone replacement doses (11).

Prednisone administered orally once to twice daily can be used as an alternative to hydrocortisone when growth is complete. Use of dexamethasone is not recommended because of difficulties with dose titration and high risk for cushingoid side effects (94).

MINERALOCORTICOID REPLACEMENT

Mineralocorticoid replacement with fludrocortisone at doses 0.05-0.2 mg daily is recommended in patients with PAI and confirmed aldosterone deficiency (16). Higher doses (i.e., 0.1-0.2mg daily) are typically required during the first year of life as newborns have lower mineralocorticoid sensitivity. In addition, infants typically require salt supplementation with sodium chloride, at doses of 1-2 grams daily given in 3-4 divided doses. Treatment with sodium chloride is gradually reduced during the first couple of years of life and replaced with unrestricted salt supplementation with food as the child transitions to regular diet. Extra fluids with electrolytes are recommended when excessive sweating is anticipated, such as during vigorous exercise or hot weather.

MONITORING DURING THERAPY

Monitoring of Glucocorticoid Replacement

There is no good biomarker to assess glucocorticoid replacement. Morning ACTH concentrations are frequently elevated and attempts to normalize ACTH levels may result in overtreatment (16). Measurements of serum cortisol concentrations after hydrocortisone administration is not routinely done. Monitoring of therapy, therefore, relies primarily on clinical assessment. Morning nausea with poor appetite and weight loss can all be signs of inadequate treatment. Children should be questioned about their daily activities and energy during the day (i.e., need for napping after school), and the information should be considered when titrating medication doses. Growth velocity and weight gain are also sensitive clinical indicators. Specifically, overtreatment with glucocorticoids is associated with poor linear growth and an increase in BMI. Appropriate progression through puberty is reassuring.

Monitoring of Mineralocorticoid Replacement

As with glucocorticoids, mineralocorticoid replacement is also assessed clinically starting with questions related to salt craving and measurements of standing blood pressure and heart rate (16). Fatigue and poor growth can also be signs of inadequate mineralocorticoid replacement (95).

Measurements of serum potassium and plasma renin (level or activity) can be used for titration of fludrocortisone dose and sodium chloride supplementation (1,2,16,94). Goals are to maintain serum potassium concentrations in the normal range and plasma renin in the upper range of normal. Orthostatic hypotension, high renin and hyperkalemia indicate undertreatment. On the contrary, low or suppressed plasma renin along with a low serum potassium concentration are signs of mineralocorticoid overtreatment. In such cases, blood pressure needs to be assessed to ensure that the child does not experience hypertension, and fludrocortisone and sodium chloride doses adjusted. In the case of hypertension, one must also remember that hydrocortisone has mineralocorticoid activity and high replacement doses can contribute to blood pressure elevations. Thus, hydrocortisone doses need to be assessed and titrated as needed.

Sick Day Management and Stress Dosing

Endogenous cortisol secretion increases during acute illness, anesthesia, surgery, or trauma. Hence, individuals with AI require an increase in their glucocorticoid doses during times of physiological stress to avoid adrenal crisis.

The stress dose regimens for children are largely empirical or consensus driven and may vary across practices. With lack of good quality evidence, many pediatric recommendations are adopted from adult literature (116). As an overarching principle, stress dose management errs on the side of overtreatment to avoid a potentially life-threatening adrenal crisis.

For management purposes, the stressor is frequently referred to as “moderate” or “severe”. Severe stress refers to major illness (e.g., sepsis), that requires hospitalization, major surgical procedures that require general anesthesia (e.g., abdominal surgery), or severe trauma. Moderate stress refers to an acute illness that can be managed at home or with minor surgery (e.g., a dental procedure) (table 2).

Table 2. Summary of Stress Dose Management of a Child with Adrenal Insufficiency: Indications and Doses
	Indications	Hydrocortisone dose		Comments
Adrenal Crisis	Vomiting, lethargy, hemodynamic instability	Age 0-24 months	25mg	♦ IVF resuscitation with 0.9% sodium chloride/5% glucose ♦ Continue with severe stress dose coverage.
		Age 2yrs - 10 yrs	50mg
		Age >10 years	100mg IM/SQ
Severe stress	Surgery, sepsis, major trauma, lethargy, repeated vomiting	100mg/m²/day divided 6 hours IV/IM Max 50mg every 6 hours		♦ Wean according to clinical status/improvement. ♦ Switch to moderate stress dosing usually when able to tolerate po.
Moderate stress	Fever >38.3^oC (101^oF) Significant trauma (i.e., broken bone), Minor surgery requiring anesthesia (e.g., dental procedures), seizures, intense exercise (i.e., marathon)	30-50 mg/m²/day divided 8 hours orally		♦ Continue stress dose coverage for up to 24 hours after stress resolves.
Prednisone can be used as an alternative if hydrocortisone is not available. Dexamethasone is the least preferable option. Additional indications: Minor cold, school exams = no need for stress dosing. Medical procedure with local anesthesia = moderate stress dose orally once prior to procedure.

MANAGEMENT OF ADRENAL CRISIS

Adrenal crisis is characterized by hypotension and volume depletion. Hypoglycemia is frequent in children. In cases of combined glucocorticoid and mineralocorticoid defects, there is additional urinary sodium loss resulting in electrolyte abnormalities (i.e., hyponatremia, hyperkalemia, acidosis, and elevated serum urea). The cornerstones of therapy are glucocorticoid replacement and fluid resuscitation.

Children with suspected adrenal crisis should immediately receive a bolus dose of hydrocortisone IM or IV (Table 2). For emergencies, a dose based on age can be used, such as 25mg, 50mg and 100mg for children <2 years, 1-10 years, and older than 10 years, respectively. The child should then continue receiving stress dose coverage with parenteral hydrocortisone at doses of 100mg/m²/day given every 6 hours (maximum dose 50mg every 6 hours) until their condition improves. If hydrocortisone is not available, prednisone (20mg/m²/day) can be used as an alternative, while dexamethasone (4mg/m²/day) is the least preferable glucocorticoid option, related to its metabolic consequences as well as lack of mineralocorticoid properties.

Fluid resuscitation can start with a bolus of 0.9% sodium chloride at 10 mL/kg. In the case of hypoglycemia, normal saline with 5% glucose can be used. Fluids should continue based on patient’s needs. Hyperkalemia can be severe at the onset of adrenal crisis but improves rapidly with parenteral glucocorticoid and fluid management. Additional therapies to lower serum potassium concentrations can be considered (i.e., IV insulin and glucose, IV calcium gluconate or cation exchange resins) if severe hyperkalemia persists.

MANAGEMENT OF SEVERE STRESS

It entails parenteral administration of hydrocortisone at 100mg/m²/day given every 6 hours (maximum dose 50mg every 6 hours) (116) (Table 2) Doses can be tapered rapidly and based on clinical improvement to the established daily glucocorticoid regimen. For children recovering from surgery, hydrocortisone can usually be changed to oral sick-day doses once they are stable and can tolerate oral fluids and diet. Children with PAI do not require fludrocortisone during severe stress coverage, as hydrocortisone also has mineralocorticoid activity. Fludrocortisone must be added back to their treatment plan as hydrocortisone is weaned down to approximately 50-60mg/m²/day.

MANAGEMENT OF MODERATE STRESS OR HOME SICK DAY RULES

Families are advised to increase their child’s daily glucocorticoid dose if they experience an intercurrent illness, such as fever or diarrhea (Table 2). Hydrocortisone doses that provide stress dose coverage for such situations (i.e. moderate stress) are in the range of 30-50mg/m²/day given every 6 or 8 hours (116). A frequent instruction to patients and families is to double or triple the daily hydrocortisone dose. This approach may lead to undertreatment if the daily dose is small. Calculation of a specific moderate stress dose based on the child’s body surface area (BSA) is preferable as it provides a more precise dosing.

Stress dose coverage is not recommended for minor upper respiratory viral infections, increased schoolwork, emotional stress, or intense exercise of brief duration. Although there is no specific guideline, some medical providers recommend using stress doses during prolonged intense training, such as a marathon.

Patients and families should undergo in-depth training around stress dose management. Instructions should include indications for moderate stress glucocorticoid administration and appropriate doses and training on using hydrocortisone as an emergency injection IM in case of vomiting, severe trauma, or impending adrenal crisis. They should be provided with a medical letter or card that documents stress dose instructions and contact information of medical providers and caregivers. Children should wear a medical alert bracelet and carry emergency supplies of oral glucocorticoids and emergency injectable hydrocortisone.

Glucocorticoid-Induced Adrenal Insufficiency: Wean and Recovery of Adrenal Function

GLUCOCORTICOID TAPERING

There is no evidence to support a specific approach to GC taper. For individuals on chronic glucocorticoid treatment (i.e., >3-4 weeks) at supraphysiologic doses for an underlying disease (e.g. inflammatory or immune disorder), glucocorticoid taper should be done at a rate dictated by the underlying condition in order to maintain disease remission (58).

Glucocorticoid withdrawal syndrome has been described primarily in adults during tapering of supraphysiologic doses or after successful treatment of Cushing (117). The syndrome can mimic signs of adrenal insufficiency (i.e., fatigue, muscle aches, nausea, abdominal discomfort, weight loss, mood swings, irritability) but is not related to untreated adrenal insufficiency, as the glucocorticoid daily dose is still supraphysiologic (117). The underlying mechanism is incompletely understood and likely involves cytokine and prostaglandin upregulation as cortisol concentrations decline. Should glucocorticoid withdrawal syndrome be suspected tapering down to physiologic doses can be done at a slower pace (58).

Once physiologic glucocorticoid doses have been achieved, a slower taper below physiologic doses have been suggested (58). The tapering aims to alleviate symptoms of adrenal insufficiency while allowing for HPA axis recovery. For those on long-acting glucocorticoids (e.g., dexamethasone), it is suggested to switch to hydrocortisone to ease with titration at small doses and faster recovery of the adrenal axis. Weaning below physiologic doses is done mostly empirically since there is no evidence about the best practice protocol. It is our practice to wean from physiologic replacement to off glucocorticoids in four-to- five steps by reducing the dose by 20-25% (Table 3). We follow a twice daily hydrocortisone/ prednisone regimen and cut down initially the evening dose to allow for faster HPA axis recovery. Children should remain on a stress dose plan until there is evidence of recovery of adrenal function.

Table 3. Glucocorticoid Induced Adrenal Insufficiency: Proposed Wean From Physiologic Replacement Doses to Off Glucocorticoids
Length of GC exposure	<4 weeks	4-12 weeks	>12 weeks
Hydrocortisone (given twice daily)	No wean	10mg/m²/day x 4 days	10mg/m²/day x 7 days
		8mg/m²/day x 4 days	8mg/m²/day x 7 days
		6mg/m²/day x 4 days	6mg/m²/day x 7 days
		4mg/m²/day x 4 days	4mg/m²/day x 7 days
		stop	stop
Prednisone (Given twice daily)	No wean	4mg/m²/day x 4 days	4mg/m²/day x 7 days
		3mg/m²/day x 4 days	3mg/m²/day x 7 days
		2mg/m²/day x 4 days	2mg/m²/day x 7 days
		1mg/m²/day x 4 days	1mg/m²/day x 7 days
		Stop	Stop

ASSESSMENT AND RECOVERY OF ADRENAL FUNCTION

Adrenal function recovers once supraphysiologic doses of glucocorticoids are discontinued. The time to recovery, however, is variable and dependent on length and potency of the glucocorticoid that was used (58). Assessment of adrenal function can be done with the measurement of morning cortisol concentrations. Because hydrocortisone interferes in cortisol measurements, testing should be at least 18–24 hours after the last dose. Adult data support a morning cortisol value above 12 mcg/dL as indicative of normal adrenal function, while values less than 5 mcg/dL suggest suppression of the HPA axis. These cortisol cut-offs are dependent on the cortisol assay that is used. An ACTH stimulation test can be considered for intermediate cortisol values (i.e. 5-12mcg/dL). An alternative approach can be to continue stress dose steroid coverage and repeat a measurement of morning cortisol after few weeks and until recovery of the axis is documented.

USEFUL LINKS/GUIDELINES

Diagnosis and Treatment of Primary Adrenal Insufficiency: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 https://pubmed.ncbi.nlm.nih.gov/26760044/
European Society of Endocrinology and Endocrine Society Joint Clinical Guideline: Diagnosis and Therapy of Glucocorticoid-induced Adrenal Insufficiency. J Clin Endocrinol Metab. 2024 PMC11180513.https://pubmed.ncbi.nlm.nih.gov/38724043/
Hormonal Replacement in Hypopituitarism in Adults: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 https://pubmed.ncbi.nlm.nih.gov/27736313/
Emergency and perioperative management of adrenal insufficiency in children and young people: British Society for Paediatric Endocrinology and Diabetes consensus guidance. Arch Dis Child. 2023 https://pubmed.ncbi.nlm.nih.gov/37045585/

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Infections Of The Hypothalamic-Pituitary Region

ABSTRACT

Infections of the hypothalamic-pituitary region are rare lesions, accounting for less than 1% of all pituitary lesions. The clinical diagnosis of these infections can be difficult due to the nonspecific nature of the disease (in many patients without symptoms of infection) and may be misdiagnosed as other pituitary lesions. The risk factors for infections of the hypothalamic-pituitary region are meningitis, paranasal sinusitis, head surgery, and immunocompromised host (diabetes mellitus, Cushing’s syndrome, HIV infections, solid organ transplantation, malignancy). Infections can develop in a normal pituitary gland or in pre-existing pituitary lesions (adenoma, Rathke´s cleft cyst, craniopharyngioma). There are several modes of dissemination of the infection to the hypothalamic-pituitary region: hematogenous, iatrogenic (after neurosurgical procedures), and spread from paranasal or nasal cavity (through venous channels of the sphenoid bone). Hypothalamic-pituitary infections most commonly present with visual disturbances and headache, in some cases with fever and leukocytosis. A significant proportion of patients develop hypothalamic-pituitary dysfunction during the acute phase of the disease or months and years after successful antimicrobial therapy . Diagnosis can be challenging and the hypothalamic-pituitary infection with formation of abscess or granuloma may be misdiagnosed as a pituitary tumor. Transsphenoidal drainage followed by antibiotics, antimycotics, or anti-tuberculous drugs are usually efficient in successful treatment of these patients.

INTRODUCTION

Infections of the hypothalamic-pituitary region are rare and commonly described in case reports or small case series. These infections include bacterial infections (pituitary abscess), tuberculosis, fungal, viral, and parasitic infections (Table 1). An infection in the hypothalamic-pituitary region may present as a sella/suprasellar mass and may be misinterpreted as a pituitary tumor. Also, these infections may cause hypopituitarism and be misdiagnosed as post-encephalitic syndrome (1-4).

Table 1. Infectious Agents which Cause Hypothalamic-Pituitary infections
BACTERIA
· Gram-positive cocci (Staphylococcus, Streptococcus)
· Gram-negative cocci (Neisseria, Esherichia coli , Pseudomonas, Brucella)
· Spirochete (Treponema pallidum, Leptospira Interrogans)
· Mycobacteria (M. Tuberculosis)
VIRUS
· Herpes simplex virus
· Varicella zoster virus
· Cytomegalovirus
· Tick-borne
· Hantaan (Hantan) virus
· Enterovirus
· Neuroborreliosis
· SARS-CoV-2 virus
FUNGI
· Candida
· Aspergillus
PARASITES
· Toxoplasma gondii, Echinococcus, Taenia solium

Infections of the hypothalamic-pituitary region may be primary (without an identifiable source) or secondary in origin (3, 5). The more common is a primary pituitary infection, which occurs in previously healthy normal pituitary glands. Secondary pituitary infections occur in patients with a pre-existing lesion in the pituitary region (pituitary adenoma, Rathke´s cleft cyst, craniopharyngioma, or prior pituitary surgery).

There are several sources of infections in the hypothalamic-pituitary region (Table 2). Dissemination from the sphenoid sinus to the pituitary is possible by direct contact and through shared venous drainage.

Table 2. Sources of Infections Spreading to the Hypothalamic-Pituitary Region
Spread	Comments
· Hematogenous spread	In immunocompromised host
· Direct extension from adjacent anatomical sites	Meningeal infection, sphenoid sinus, cavernous sinus, skull
· Previous infectious diseases of the CNS
· Iatrogenic	Surgical intervention in sellar and suprasellar region, tooth extraction

Infections in the hypothalamic-pituitary region are rare and several predisposing factors have been identified (2) (Table 3).

Table 3. Predisposing Factors for Hypothalamic-Pituitary Infections
· Diabetes mellitus
· Tuberculosis
· Solid organ transplantation (renal, liver, etc.)
· Human immunodeficiency virus (HIV) infection
· Non-Hodgkin lymphoma
· Chemotherapy
· Cushing´s syndrome
· Previous pituitary surgery
· Immunosuppressive therapy

Infections of the hypothalamic-pituitary region may present with neurological signs and symptoms and signs of neuroendocrine dysfunction (Table 4).

Table 4. Clinical Features of Hypothalamic-Pituitary Infections
NEUROLOGICAL SYMPTOMS	ENDOCRINE DYSFUNCTION
Headache	Hyponatremia
Visual disturbances	Hypopituitarism
Cranial neuropathy (III, IV, VI)	Hypogonadotropic hypogonadism
	Isolated ACTH deficiency
	Hyperprolactinemia
	Central diabetes insipidus

HYPOTHALAMIC-PITUITARY BACTERIAL INFECTIONS

Pituitary Abscess

Pituitary abscesses are rare pituitary lesions accounting for less than 1% of all pituitary lesions (6, 7). The first case of a pituitary abscess was described in 1848 and since then it has been mostly described in case reports or small series. In two-thirds of patients, pituitary abscesses occur in previously healthy normal glands (primary pituitary abscess) (8). In other patients, there is a preexisting lesion in the pituitary region, such as a pituitary adenoma, Rathke´s cleft cyst, granulomatous hypophysitis, or craniopharyngioma or prior pituitary surgery (secondary pituitary abscess) (5, 9-12). The infection can be caused by hematogenous dissemination or by direct extension from surrounding structures (meningitis, sphenoid sinusitis, cavernous sinus thrombophlebitis) (Table 2). Pituitary surgery and immunocompromised condition are also risk factors for pituitary abscesses (Table 3).

According to the clinical presentation and duration of the disease, pituitary abscesses can be acute, subacute (the disease course less than 1 month), or chronic (disease course longer than 1 month) (13). Infective manifestations (fever, leukocytosis, meningism) have been reported in patients with acute and subacute pituitary abscesses, while chronic pituitary abscesses have a more indolent course.

Many patients with pituitary abscess were misdiagnosed as having a pituitary adenoma, pituitary adenoma with apoplexy, or Rathke´s cleft cyst prior to surgery (14). The diagnosis of this potentially life-threatening disease is based on intraoperative detection of pus and postoperative histopathological analysis. Clinically, pituitary abscesses usually present with neurological signs and symptoms (headache, visual disturbances), signs of neuroendocrine dysfunction (anterior hypopituitarism, AVP deficiency) and signs and symptoms related to infections (fever, leukocytosis) (5, 8, 14). The largest series of primary pituitary abscesses (84 patients) during a 20-year period reported asthenia as the most common clinical presentation (75%), followed with visual impairment (71%), and headache (50%) (6).

Compared to patients with a pituitary adenoma who rarely have neuroendocrine dysfunction, most patients with pituitary abscesses have hypopituitarism (8, 9, 13). The large case series of 66 pituitary abscesses reported anterior pituitary hypopituitarism in 81.8% of patients, while AVP deficiency was diagnosed in 47.9% of patients (8). Nine percent of patients (9.3%) had isolated hypogonadism, 3.7% had isolated ACTH deficiency, 1.8% had isolated hypothyroidism, and 1.8% had combined hypogonadism and ACTH deficiency (8). The possible source of the pituitary infection was found in 14 out of 66 patients (sepsis, sinusitis, pulmonary tuberculosis) (8). Recently published the largest series of 84 patients with primary pituitary abscesses confirmed the high incidence (73%) of preoperative neuroendocrine dysfunction in patients with pituitary abscess: panhypopituitarism in 46%, isolated corticotropic insufficiency in 13%, isolated thyrotropic insufficiency in 10%, isolated gonadotropic insufficiency in 8%, and combined two pituitary axes insufficiencies in 22% (6).

On MRI, pituitary abscesses present as a sella masses, hypointense or isointense on T1-weighted imaging, hyperintense or isointense on T2-weighed imaging, with typical rim enhancement after gadolinium injection, mimicking apoplexy of the pituitary adenoma or other cystic sella lesions (7-9, 13, 15-17; Fig. 1). Diffusion-weighted imaging (DWI) sequences of pituitary abscesses often demonstrate high signal intensity with a reduction in the apparent diffusion coefficient, different from necrotic brain tumors (18).

Figure. 1. Pituitary abscess: gadolinium-enhanced T1-weighted MRI scan (sagittal view) shows sellar and suprasellar mass with peripheral contrast enhancement.

Neuroimaging with nuclear medicine investigations (18-FDG PET scan, labelled leukocyte scintigraphy) could increase the preoperative diagnostic rate in challenging patients with pituitary abscess (6).

The majority of patients are treated with transsphenoidal surgery, rarely with a transcranial approach (8). Intraoperatively, pus is found in the sella (19, 20). In patients with secondary pituitary abscesses, the sphenoid sinus is the most common site of extrasellar invasion (5).

On histopathological analysis, there is evidence of acute or chronic inflammation, while Gram staining and bacterial cultures in some cases may identify the infecting pathogen. In most cases, the etiological agents cannot be isolated (6, 12). In the two largest studies on pituitary abscesses, positive results on gram staining or bacterial cultures were found in only 19.7% and 25% of patients, respectively (6, 8). The most prevalent organisms are Gram-positive cocci (Staphylococcus Aureus and Streptococcus species), but also Gram-negative bacteria (Neisseria, Esherichia coli, Pseudomonas, Brucella) (5, 6, 9, 21).

Patients with bacterial pituitary abscesses are treated with intravenous and oral antibiotics for three to six weeks to prevent the recurrence of the pituitary abscesses, but in some cases, reoperation was required. In rare cases, AVP deficiency and hypopituitarism are reversible, occasionally followed by secondary empty sella (22). In most cases, neuroendocrine dysfunction and AVP deficiency are irreversible findings (6, 13). The preoperative diagnosis of pituitary abscess represented a protective factor for pituitary function recovery (in 23% of patients) (6).

Although pituitary abscesses are more indolent than other intracranial abscesses, secondary pituitary abscesses in patients with pituitary adenomas (not in Rathke´s cleft cyst) is associated with high mortality rate (26%) due to the dissemination of the infection or meningitis (5). In patients with infected Rathke´s cleft cyst, clinical manifestations are commonly subacute, without septic symptoms (9, 11).

Recently published the largest systematic review of 488 cases of pituitary abscess examined presentation, radiological findings, endocrinological abnormalities, treatment, and mortality of these patients (23). The most common symptoms were headache (76%) and visual field loss. The median time from onset of symptoms to presentation was 120 days. Symptoms and biochemical markers of infection were absent in 57% of cases. The appearance of hypointensity on T1 weighted images and hyperintensity on T2 weighted images, as well as peripheral contrast enhancement of the pituitary on MRI were the most common radiological findings. Fifty-five percent of patients had negative culture results. Endocrinological abnormalities were present in 84.5% of cases (panhypopituitarism in 41%, AVP deficiency in 25%) and persisted in over half of cases. The mortality rate was 5.1%, with delayed presentation increasing risk of mortality (23).

Hypopituitarism Caused by Treponema Pallidum Infection (Syphilis)

Syphilis is a sexually transmitted chronic bacterial infection caused by Treponema Pallidum which progresses over years through a series of clinical stages. Syphilis is a well-recognized cause of hypopituitarism, with granulomatous hypophysitis (noncaseating giant cell granuloma), syphilitic gumma in sella region, or congenital syphilis causing hypothalamic-pituitary dysfunction. The first cases of hypopituitarism caused by syphilis were described almost 70 years ago, mostly in postmortem cases (24). The use of penicillin caused a decline in syphilis presentations and late complications, including congenital syphilis. Nowadays, the incidence of syphilis has been rising again and this sexually transmitted disease should be considered again in the differential diagnosis of neurological, psychiatric, and endocrine cases in high-income countries in risk groups (HIV positive patients, men-men relationships, crack cocaine users, and among intravenous drug users) (25, 26). Spirochete Treponema Pallidum is also described as a cause of hypophysitis and pituitary gland enlargement with hypopituitarism, mostly in immunocompromised patients (HIV-infected patients) with syphilitic meningitis (27). Syphilis may cause a sella mass with suprasellar extension mimicking a pituitary tumor and causing severe headache and hypopituitarism (28). The diagnosis is confirmed by positive treponemal antibody or by detection of Treponema Pallidum by immunohistochemistry or PCR on the resected pituitary. This disorder is treated with antibiotics.

Hypopituitarism Caused by Leptospira Interrogans Infection (Sy Weil)

Leptospirosis is a common tropical febrile, zoonotic infectious disease caused by spirochete Leptospira Interrogans. The bacteria are spread through the urine of infected animals (cattle, pigs, horses, dogs, rodents, wild animals). Humans can become infected through contact with urine or other body fluids from infected animals or through contact with water, soil, or food contaminated with the urine of infected animals. This disease presents with hepatorenal syndrome and systemic hemorrhagic manifestations. The first case of pituitary apoplexy and panhypopituitarism caused by leptospirosis in a 56-year-old male with type 2 diabetes mellitus was recently published (29). The patient developed fever, nausea, vomiting and acute kidney injury. Leptospirosis was diagnosed by positive leptospira antibody test, and he started treatment with antibiotics. After five days of admission, he developed signs and symptoms of pituitary apoplexy. A brain MRI scan was consistent with apoplexy in a pituitary adenoma (the mass showed T2W hyper intensity and TIW isointensity with hypo intense areas which suggested hemorrhage). The patient developed hypopituitarism and was replaced with glucocorticoids and thyroid hormones. The follow-up MRI scan showed resolution of the hemorrhagic focus and regression of the pituitary adenoma. The proposed mechanism of pituitary apoplexy is platelet dysfunction (caused by uremia and directly by leptospira) and non-inflammatory vasculopathy (increased vascular permeability due to disruption of endothelial cell-cell junctions, cell retraction and opening of intercellular gaps) (29). Leptospirosis clinically presents very similar to another zoonosis, hantavirus infection, is more common worldwide and serology and PCR are necessary to distinguish between these two diseases (30).

Hypothalamic-Pituitary Dysfunction Following Bacterial CNS Infections

Hypothalamic-pituitary dysfunction is a well-recognized complication of acute infectious diseases of the central nervous system (meningitis and encephalitis) and may occur in the acute phase or in the late stage of these diseases (1, 4, 31, 32). The clinical spectrum of neuroendocrine dysfunction may range from an isolated pituitary hormone deficiency to panhypopituitarism. Endocrine dysfunction in the acute phase of meningitis may return to normal after the acute period or be irreversible (32). The most common deficit is isolated GH deficiency diagnosed 6-48 months after the infection, reported at a rate of 28.6% (31).

Hypopituitarism following acute viral or bacterial meningitis in children is not as common as in adulthood (33, 34). The GH neurosecretory dysfunction (low IGF1 with normal GH response in clonidine test) was found in 3 out of 37 children tested at least 6 months following the diagnosis of bacterial meningitis (34). There are rare case reports on hypopituitarism during acute meningitis caused by Streptococcus Group B or sepsis caused by Salmonella enteritidis in a neonatal period (35, 36).

The pathophysiological mechanism responsible for hypothalamic-pituitary dysfunction following acute meningitis is not fully understood. In some patients, anti-pituitary and anti-hypothalamus antibodies are detected (37). It is proposed that acute infection provokes an autoimmune process and may cause axonal injury with consequent neuroendocrine dysfunction (38).

Idiopathic Granulomatous Inflammation of the Cavernous Sinus - the Tolosa-Hunt Syndrome

Tolosa-Hunt syndrome is defined as an idiopathic granulomatous inflammation of the cavernous sinus, therefore not infectious in origin, with variable extension into the superior orbital fissure/orbital apex, usually unilateral. The diagnosis is made by exclusion of other more common causes of cavernous sinus lesions (thrombosis, tumors, fungal infections, systemic granulomatous diseases-sarcoidosis, tuberculosis, Wegener´s granulomatosis) (39). In less than 5% of cases it can be bilateral, mimicking a pituitary adenoma in imaging studies (40). The etiology of Tolosa-Hunt syndrome is not fully understood. The disease is characterized by nonspecific granulomatous inflammation with infiltration of lymphocytes and plasmocytes. The patient presents with severe unilateral orbital pain and ipsilateral ocular motor neuropathy. The paralysis of one or more cranial nerves passing through the cavernous sinus (III, IV, VI) develops with orbital pain after less than 2 weeks. The signs of infection (fever, leukocytosis) are usually present. Granulomatous inflammation develops within the cavernous sinus causing acute throbbing orbital pain and disordered eye movement. Brain MRI scan demonstrate the inflammation in the cavernous sinus, orbital apex, and rarely in the sella. MR venography of the brain is important to exclude cavernous sinus thrombosis. Treatment consists of high dose corticosteroids and antibiotics. Refractory and steroid-intolerant cases may be treated with immunosuppressants (Methotrexate or Azathioprine) and gamma knife radiotherapy (41). Periorbital pain intensity is rapidly decreasing and resolving within 72h, while the resolution of ophthalmoplegia improves gradually and takes a longer time to resolve (several weeks) (42). If the patient is not responding to standard therapy, biopsy of the lesion is necessary to exclude other diseases, such as lymphoma.

Hypothalamic-Pituitary Tuberculosis

The incidence of tuberculosis is rising not only in developing countries, but also in developed countries, especially given the increasing population migration. Mortality has begun to increase after years of decline and since 2018 more than 7 million people have died of tuberculosis (43). Extrapulmonary tuberculosis may affect the brain causing tuberculous meningitis and tuberculoma of the central nervous cases. Tuberculous meningitis has a tendency to affect basal parts of the brain from where it can spread to the sella region. In rare cases, CNS tuberculosis may present as tuberculous hypophysitis or sella/suprasellar tuberculoma mimicking a pituitary adenoma or pituitary apoplexy (44-48). It may occur in the absence of systemic tuberculosis, but the majority of patients have a past history of pulmonary tuberculosis or tuberculosis of other organs (spine). Tuberculosis may affect the hypothalamus, pituitary, paranasal sinuses (sphenoid sinus), or tuberculoma may be located only in the pituitary stalk. Hypothalamic-pituitary dysfunction and AVP deficiency during the acute phase or years after recovery from acute tuberculous meningitis suggests a more destructive and more extensive hypothalamic and pituitary damage compared to other causes of acute viral and bacterial meningitis.

A significant proportion of patients with sella tuberculoma or tuberculous meningitis develop hypothalamic-pituitary dysfunction. In 18 cases of histologically proved sella tuberculoma (5 of them with past history of tuberculosis), 5 patients had hypopituitarism and 3 had hyperprolactinemia due to pituitary stalk compression (44).

In patients with tuberculous meningitis half of them developed neuroendocrine dysfunction: hyperprolactinemia (23-50% of patients), hypocortisolism (13-43%), hypothyroidism (17-31%), hypogonadism (34%), SIADH (10%) (4, 49, 50) during the acute phase. Multiple hormonal axes were affected in 23.5% of patients (49, 50). In young adult patients who survived tuberculous meningitis in childhood and were tested several years after recovery, hypopituitarism was diagnosed in 20% of patients (51). This is a consequence of fibrosis, gliosis, and calcification in the hypothalamus and pituitary after recovery of active tuberculous brain infection. In rare cases, pituitary function recovered after successful treatment with anti-tuberculous drugs (52).

The diagnosis of sella tuberculoma is a challenge, especially in cases without systemic tuberculosis. The signs and symptoms are nonspecific: fever, neurological abnormalities (headache, visual disturbances), and neuroendocrine dysfunction (44, 53). Sellar tuberculoma on MRI scans presents as thickening of the pituitary stalk or abnormal enhancement pattern of the sella lesion (54) (Fig. 2).

Figure 2. Tubercular hypophysitis: sellar MRI scan (coronal view) shows stalk thickening.

If the correct diagnosis is established, anti-tuberculosis drugs are effective and surgery is not indicated for tuberculous hypophysitis. With surgery the histological examination shows granulomas with central caseous necrosis surrounded by giant Langhans cells. In cases in whom Ziehl-Nielsen staining for acid fast bacilli and the culture on Lowenstein-Jensen media are negative, PCR for detection of mycobacterial DNA in tissue or CSF may help.

HYPOTHALAMIC-PITUITARY FUNGAL INFECTIONS

Hypothalamic-pituitary fungal infections are extremely rare and occur usually in immunocompromised patients (diabetes mellitus, granulocytopenia, solid organ transplantation). There are only few case reports of Candida and Aspergillus sella abscesses and reviews of fungal sellar abscesses (55-61). Aspergillus is a ubiquitous saprophitic fungus found in the nasal mucosa of healthy people and patients with chronic sinusitis. This fungus may cause CNS infection, such as meningitis, encephalitis, brain abscess, subdural abscess, pituitary abscess, and mycotic arteritis with thrombosis and aneurysm (61-63). Fungal sella abscesses have a nonspecific presentation, with neurological signs and symptoms (headache, visual disturbances) and hypothalamic-pituitary dysfunction. Sella MRI images are nonspecific, with a T1W hypointense or isointense mass with rim enhancement and may be misdiagnosed as a pituitary adenoma (Fig. 3). It is proposed that a low signal on T2W images due to iron deposition may be a more specific sign of fungal abscess (58, 64; Fig. 4). Diagnosis of fungal pituitary abscess is made by histopathological finding (Grocott–Gömöri's methenamine silver stain demonstrates septate fungal hyphae), cultivation, or PCR identification of fungus DNA. A combination of transsphenoidal drainage and antifungal therapy (liposomal Amphotericin B, itraconazole, voriconazole, caspofungin, micafungin) can result in a good prognosis (55, 59, 60, 65). Endocrinopathies caused by fungal abscess have a low rate of recovery (59).

Figure 3. Fungal pituitary abscess spreading from fungal sinusitis: sellar MRI scan (coronal and sagittal views) shows a large sellar mass pushing the pituitary upwards.

Figure 4. Fungal infections in the sella: a) CT scan of the sinuses (axial view) shows a large sellar mass, erosion in sellar floor, propagation of the pathological process and opacification of the sinuses, and B) sellar MRI scan (coronal view) shows a giant hypointense lesion in the sellar region.

An unusual form of allergic fungal sinusitis which expands from the sphenoid sinus through a bone erosion to the sella in immunocompetent patient has been described (64) (Fig. 4). This patient had hyperprolactinemia (in the setting with no pituitary stalk compression), which resolved after successful transsphenoidal operation followed by anti-mycotics and corticosteroids (64). It is speculated that fungal glucans may directly stimulate glucan-specific receptors on somatomammotroph cells to stimulate prolactin secretion (66).

HYPOTHALAMIC-PITUITARY VIRAL INFECTIONS

Hypothalamic-pituitary dysfunction (hypopituitarism and cranial diabetes insipidus) may develop in the acute phase of viral infections of the CNS (meningitis and encephalitis) or in the late stage of these diseases (1, 31, 32). Infectious agents which cause CNS viral infections are listed in Table 5.

Table 5 – Infectious Agents Which Cause CNS Viral Infections
MENINGITIS	ENCEPHALITIS
Herpes virus	Tick-borne
Varicella	Herpes simplex
Enterovirus	Cytomegalovirus
	Neuroborreliosis
SARS-CoV-2 virus	SARS-CoV-2 virus

The investigation of hypothalamic-pituitary function at least 6 months after recovery from mild-to-moderate meningitis/encephalitis showed that 21% patients developed isolated corticotroph deficiency, while other neuroendocrine abnormalities or AVP deficiency were not found (1).

Hantavirus

Hemorrhagic fever with renal syndrome (HFRS), caused by Hantaviruses in the Bunyaviridae family, is an endemic zoonotic disease transmitted by rodents. There are several serotypes of these RNA viruses causing systemic infection, milder form called nephropathia endemica (Puumala) or severe form (Dobrava, Belgrade). The disease is endemic in Europe (Balkans, Finland, Germany) and Asia (Korea), where several outbreaks have been recorded. Farmers and solders are exposed to the virus by inhalation of infected rodent urine, feces, or saliva. Hantavirus infiltrates the vascular system causing increased capillary permeability, renal failure, thrombocytopenia, hemorrhage, fever, hypotension, and shock. The mortality rate is 6.6%. Autopsy findings reported a slightly enlarged pituitary with ischemia/infarction, hemorrhage, and necrosis (67-69). Direct viral invasion was confirmed in the pituitary causing viral hypophysitis (69). Hypothalamic-pituitary dysfunction has been reported during the acute phase of the disease or after long-term follow-up (70-79). A milder form of HFRS infection caused by Puumala virus (nephropathia epidemica) is associated with a lower incidence of hypopituitarism (80). It is speculated that in some patients with no signs of hemorrhage in the sella, hantavirus may cause autoimmune hypophysitis and hypopituitarism (81). Sellar MRI imaging in hypopituitary patients reveals an edematous pituitary gland or increased signal intensity in the pituitary due to hemorrhage during the acute phase, while pituitary atrophy and secondary empty sella develops months and years after acute infection (76, 79-81) (Fig 5). A retrospective study of 60 adults who had recovered from HFRS reported that 18% of patients developed hypopituitarism (82). Ten percent of patients had a single pituitary deficit (three GH, two gonadal, and one adrenal), and 8.3% had multiple pituitary hormone deficiencies (82). In rare cases, HFRS may cause injury of the pituitary stalk or acute/subacute hemorrhage in the pituitary gland and AVP deficiency with panhypopituitarism (30, 77).

Figure. 5. Hemorrhagic fever with renal syndrome: sellar MRI scan (sagittal view) shows pituitary atrophy and secondary empty sella.

SARS-CoV-2

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as a cause of coronavirus disease 2019 (COVID-19). This virus is responsible for a variety of clinical manifestations, ranging from an asymptomatic stage to severe pulmonary disease (respiratory distress syndrome) and various extrapulmonary manifestations, including endocrine axes (83-85).

SARS-CoV-2 is a neuroinvasive virus which enters the brain through the nasopharyngeal epithelium via the olfactory nerve, passes through the blood-brain barrier, or enters the brain through the median eminence where this barrier is absent (86). Neurons, glial cells, and cerebrovascular endothelia cells, as well as hypothalamus and pituitary cells express an angiotensin-converting enzyme 2 (ACE2) receptor responsible for the entry of the virus into these cells which induce neuroinflammation (86-88). SARS-CoV-2 virus is also detected in cerebrospinal fluid of patients with encephalitis caused by COVID-19 and in pituitary tissues from patients who died from SARS (89, 90). SARS-CoV-2 is responsible for a variety of neurological complications, including headache, anosmia, confusion, ataxia, neuropathic pain, seizures, and delirium (89).

There are also indirect systemic effects of COVID-19 virus: an altered immune response (cytokine storm), infection-induced thrombocytopenia, platelet dysfunction, coagulopathy (hypercoagulable state), endothelitis, and endothelial dysfunction (91). All these direct and indirect systemic effects of COVID-19 virus may be responsible for ischemic and hemorrhagic vascular syndromes, affecting also the hypothalamo-pituitary region. Another possible indirect effect of COVID-19 may be mediated by cytokines that can trigger hypothalamo-pituitary inflammation with consequent neuroendocrine dysfunction (92). Also, it has been shown that SARS-CoV-2 express an amino-acid sequence that mimics human ACTH and induces the production of autoantibodies against ACTH causing cortisol insufficiency and inadequate adrenal response to the stress (83).

SARS-CoV-2 infection may involve the hypothalamo-pituitary axis causing pituitary apoplexy, hypophysitis, and hyponatremia (83, 85, 91, 93-97).

There are nine case reports of pituitary adenoma (6 males/3 females, between 27 and 56 years of age, eight with pituitary macroadenoma) and two series of three patients with pituitary adenomas, complicated by apoplexy during COVID-19 infection (93-102). In one patient this occurred during the third trimester of pregnancy. Four patients had transsphenoidal surgery and recovered, one patient had transcranial resection, three patients were conservatively managed, and one patient died 12 hours after admission. There is also a report of a 65-year-old woman with no underlying pituitary disease who developed acute pituitary apoplexy one month after the initial diagnosis of COVID-19 (103). She developed anterior hypopituitarism with no evidence of AVP deficiency. An MRI pituitary scan showed the resolution of intrapituitary hemorrhage, with normal size of the pituitary gland after six months of follow-up. A small case series reported three patients with previously unknown pituitary adenoma complicated by apoplexy during COVID-19 infection: a 54-year-old female with null cell adenoma, a 52-year-old and a 56-year-old men, both obese with hypertension, both with a lactotroph pituitary adenoma (94). In another series of three patients with pituitary macroadenoma complicated with apoplexy following a symptomatic COVID-19 infection, two of these patients’ developed symptoms of pituitary apoplexy days following the viral infection, whereas the third patient developed pituitary apoplexy after a 2-month period (96).

In some patients COVID-19 caused an acute lymphocytic hypophysitis with temporal evolution of symptoms, suggesting an immune-mediated parainfectious pattern of disease (104, 105). An 18-year-old previously healthy girl with a history of symptomatic COVID-19 three weeks prior to the acute onset of headache and dizziness, presented with diffuse thickening and enlargement of the infundibulum with homogenous contrast enhancement of the pituitary (104). She was treated with glucocorticoids with a significant clinical improvement on day 3 and complete resolution of MRI finding on day 5. A similar case is a 16-year-old girl who presented with headaches, polyuria/polydipsic syndrome, and impaired visual acuity three weeks after COVID-19 infection (105). She had pituitary enlargement on MRI, was treated with methylprednisolone and improved on day 5. Such a rapid headache resolution after steroid treatment suggests a transitory acute hypophysitis, an immune-mediated process, triggered by viral infection. It has been hypothesized that SARS-CoV-2 may induce hypothalamo-pituitary autoimmunity, with positive anti-hypothalamus (AHA) and anti-pituitary (APA) antibodies (106). The current results in the literature about antigens which are targets of AHA and APA are still contradictory (83). It is possible that patients at high-risk for such a complication are carriers of specific HLA alleles. The human leukocyte antigen (HLA) complex has a central role in the recognition and presentation of viral antigens to immune system. HLA class I molecules mediates innate defense strategies against viral infection. HLA-c 04:01, DRB1 08, DQB1 06 carriers have an increased risk of a severe clinical course of COVID-19 and generate a robust response and susceptibility to autoimmune diseases (107-110).

Even these two acute pituitary disorders (pituitary apoplexy with transition to acute hypophysitis) can occur in patients with COVID-19 and preexisting unknown pituitary adenoma (97).

Several cases of AVP deficiency as a late complication of COVID-19 infection were published (111-114). All patients (2 males/2 females, 28-60 years of age) suddenly developed polyuria, nocturia, and polydipsia four to eight weeks after the diagnosis of COVID-19 infection. In one of the published cases, a sellar MRI scan showed an enlarged pituitary with an absent posterior pituitary bright spot on T1W images associated with thickening of the pituitary stalk of 3.5 mm suggesting infundibulo-neurohypophysitis (113). Other pituitary hormone evaluations were normal. They were treated with oral desmopressin.

A case of a young woman with hypothalamic amenorrhea following COVID-19, with normal appearance of the sella turcica and regular dimensions of the pituitary was reported (95). This case suggests clinicians need to follow patients for possible delayed neuroendocrine dysfunctions after COVID-19 infection.

Hyponatremia was reported in 30-60% of patients with SARS-CoV-1 infection and was associated with worse outcomes and increased mortality (115). The syndrome of inappropriate anti-diuretic hormone secretion (SIADH) was the most common reason for hyponatremia, followed by adrenal insufficiency (116).

There are also data on the association between COVID-19 vaccination and the subsequent development of pituitary diseases (85, 117-120). A recently published systematic review analyzed 23 case reports of post COVID-19 vaccination pituitary diseases: hypophysitis in 9 patients, pituitary apoplexy in 6 patients, SIADH in 5 patients, and isolated ACTH deficiency in two patients (119). Symptoms of pituitary disease typically occurred shortly (several days) after vaccine administration and the pathogenetic mechanisms potentially include molecular mimicry, vaccine adjuvants, and vaccine-induced thrombotic thrombocytopenia (83, 119). The presence of ACE2 receptors in the hypothalamo-pituitary system contributes to these post vaccinal pituitary diseases. Isolated infundibulo-neurohypophysitis and AVP deficiency or isolated ACTH deficiency may also develop several days after immunization with BNT 162b2 mRNA COVID-19 vaccine (117, 121). In some cases of panhypopituitarism and AVP deficiency due to hypophysitis after COVID-19 vaccination, hormonal secretion partially improved during follow-up (120).

Other Viruses

Cytomegalovirus, herpes simplex, varicella zoster, and enterovirus have also been described in very rare cases of central diabetes insipidus, mainly in immunocompromised patients with encephalitis (such as HIV infection, Cushing´s syndrome, lymphoma or immunosuppressive therapy) (122-126). Direct cytomegalovirus invasion and reduction in the number of AVP and oxytocin cells was confirmed in the hypothalamus (123).

HYPOTHALAMIC-PITUITARY PARASITIC INFECTIONS

Parasitic infections of the pituitary are rare and infections in the sellar region caused by Toxoplasma gondii, Echinococcus species, and Taenia solium have been reported anecdotally (4).

Toxoplasmosis is a worldwide zoonosis, caused by the protozoan parasite Toxoplasma gondii. This is one of the most common parasitic infections of warm-blooded animals and humans. Approximately one-third of humans have been exposed to T. gondii, mostly with no serious diseases, except in immunocompromised patients (HIV) and congenital toxoplasmosis. Two cases of prolactinomas with T. gondii cysts among tumor cells were reported (127). Toxoplasmosis is the most common CNS infection in immunocompromised patients (patients with HIV infection) and may cause hypopituitarism, accompanied by focal neurological deficits, headache, and fever (128, 129). The brain MRI shows lesions with significant enhancement of T2W images and peri-lesional edema, which may be misdiagnosed as intracranial metastasis. The diagnosis is based on brain biopsy with confirmed presence of T. gondii by PCR. Infection with T. gondii is treated with antimicrobial therapy and with hormone replacement therapy as needed.

The most common parasitic infestation of the brain is neurocysticercosis, caused by Taenia solium. A systematic review of 23 patients with intrasellar cysticercosis reported endocrine abnormalities (panhypopituitarism, hyperprolactinemia, AVP deficiency, and isolated hypothyroidism) in 56% of the affected population (130). These infections present with a cystic mass in the sella with hypopituitarism caused by compression, subarachnoid cysts, obstructive hydrocephalus, or neuroinflammation (ventriculitis, focal arachnoiditis) (4, 131). Neurocysticercosis may involve the pituitary stalk too (132). Transsphenoidal or transcranial operation is required for the definitive histopathological diagnosis and cure, because medical therapy with praziquantel is usually ineffective (130).

ACKNOWLEDGMENT

This study was supported by The Science Fund of the Republic of Serbia, Grant No: 7754282 — Prediction, prevention and patient’s participation in diagnosis of selected fungal infections (FI): an implementation of a novel method for obtaining tissue specimens, “FungalCaseFinder”.

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Development And Microscopic Anatomy Of The Pituitary Gland

ABSTRACT

The pituitary gland is an organ of dual origin. The anterior part (adenohypophysis) arises from embryonic buccal mucosa, whereas the posterior part (neurohypophysis) derives from neural ectoderm. Precise spatial and temporal co-ordination of transcription factor expression in both structures is critical for pituitary gland formation and the differentiation of hormone-producing cells. Disruption of this regulation, for instance by transcription factor mutation, can lead to numerous developmental disorders and disturbances in endocrine function and regulation. We provide an overview of the molecular drivers of pituitary organogenesis and illustrate the anatomy and histology of the mature pituitary gland, comprising adenohypophysis (anterior lobe), neurohypophysis (posterior lobe), pars intermedia (intermediate lobe), and infundibulum (pituitary stalk).

PITUITARY ORGANOGENESIS

The pituitary is an organ of dual origin. The anterior part (adenohypophysis) is derived from oral ectoderm and is epithelial in origin, whereas the posterior part (neurohypophysis) derives from neural ectoderm. The mixed embryological derivation of the pituitary gland requires that developmental signals from both the neural and oral ectoderm must interact temporally and spatially to control maturation of the pituitary gland and the cellular differentiation of the various hormone-producing cell types within the adenohypophysis. The expression of transcription factors that control cell lineage commitment in the developing adenohypophysis must be regulated precisely to ensure correct differentiation of hormone-producing cell types; the iterative and cumulative nature of this regulation renders it extremely sensitive to perturbation. Disruption of this process, for instance by mutation, can lead to numerous developmental disorders from congenital forms of hypopituitarism to pituitary tumors (1) (2).

Pituitary organogenesis begins during week 4 of fetal development. A thickening of cells in the oral ectoderm forms the hypophyseal placode which gives rise to Rathke’s pouch; an upward evagination of the buccal ectoderm that extends towards the neural ectoderm. At the same time, a downward extension of the ventral diencephalon forms the posterior lobe, and the two nascent lobes connect to form the bilobed structure of the adult pituitary gland. Rathke’s pouch constricts at its base and eventually separates altogether from the oral epithelium during gestational week 6-8. The cells of the anterior wall of Rathke’s pouch undergo extensive proliferation to form the anterior lobe. In humans, the posterior wall proliferates more slowly to form the vestigial intermediate lobe (3). Cell patterning and terminal differentiation occurs within the anterior lobe to form the five principal specialized endocrine cell types of the pituitary gland; the somatotrophs, adrenocorticotrophs, gonadotrophs, thyrotrophs, and lactotrophs.

TRANSCRIPTIONAL CONTROL OF PITUITARY ORGANOGENESIS

Development of the pituitary occurs broadly in three stages:

Initiation of pituitary organogenesis and formation of Rathke’s pouch (Blue in Table 1).
Evagination of Rathke’s pouch and cell proliferation (Green in Table 1).
Lineage determination and cellular differentiation (Yellow in Table 1).

Table 1. Signalling Molecules Controlling Pituitary Organogenesis and Associated Dysfunction
Developmental Stage	Factor		Function	Dysfunction	Comments	Reference
Initiation of pituitary organogenesis and formation of Rathke’s pouch	SIX homeo-domain proteins	Six1-Six6	Family of six transcriptional activators/ inhibitors Functional role difficult to determine due to redundancy and severity of mutations		Expression persists in adult pituitary; may mediate plasticity	(52) (53)
	Paired-like homeobox proteins	Hesx1	Transcriptional repressor Early marker of Rathke’s pouch Downregulation essential for endocrine cell differentiation	Mutations in patients with hypopituitarism including septo-optic dysplasia, combined pituitary hormone deficiency (CPHD) and isolated growth hormone deficiency (IGHD)	Expression activated by LIM homeodomain proteins	(54)
		Otx2	Transcription factor that regulates Hesx1	Mutations found in patients with ocular disorders (e.g. anophthalmia, microphthalmia) with or without hypopituitarism. In mice, deficiency results in craniofacial defects and pituitary gland dysmorphology, but normal pituitary cell specification		(55) (56)
		Pitx1/2/3	Interacts with various other factors to determine cell lineage	Mutations in Pitx2 (R91P) found in patients with Axenfeld-Rieger syndrome. Blocks expression of LH β and FSHβ	Expressed throughout oral ectoderm and Rathke’s pouch. Some functional redundancy, but all required for proper development Expression maintained in the adult gland	(57)
	LIM homeodomain transcription factors	Isl1	Involved in cell lineage specification	No human mutations identified. Null mice do not develop Rathke’s pouch	First LIM protein to be expressed Expressed in cells destined to become thyrotrophs	Reviewed in (6)
		Lhx3	Expression gradient required for differentiation of endocrine cell types	Heritable mutation in patients with CPHD with short, stiff neck and sensorineural hearing loss	Broad temporal and spatial expression pattern with many target genes	(58) (59)
		Lhx4	Expression gradient required for differentiation of endocrine cell types	Heterozygous mutations in patients with CPHD. Associated with pituitary hypoplasia, small sella and Arnold-Chiari malformation	Not critical for endocrine cell differentiation	(60) (61)(59)
	SOX2		Expressed throughout developing Rathke’s pouch Downregulation essential for endocrine cell differentiation	Mutations found in patients with an- or microphthalmia, hypogonadotrophic hypogonadism, and growth hormone deficiency (GHD) Both duplications and loss of function mutations associated with hypopituitarism	Some expression retained in adult pituitary, confined to pituitary progenitor/stem cells	(62) (6)
	β-catenin		Signalling activates Pitx2 expression promoting pituitary precursor proliferation. Required for Pit1 lineage determination and anterior pituitary formation	Premature activation of β-catenin results in Hesx1 repression and pituitary gland agenesis in mouse Activating mutation of β-catenin leads to pituitary progenitor proliferation, loss of Pit1 lineage cells and adamantinomatous cranyiopharyngioma	High degree of interaction with other signalling pathways e.g. Notch. Not required for cell lineage determination	(63) (7)
	Notch		Mediates lateral inhibition and cell lineage determination Activates Hes1 expression	Dysregulaiton of the pathway associated with premature corticotroph differentiation and pituitary hypoplasia in mice	Expression in the adult gland co-localises with SOX2	(64)
Migration of Rathke’s pouch cells and proliferation	Bone morphogenic proteins	BMP4	Expressed in ventral diencephalon Required for hypophyseal placode formation	Downregulation results in arrested development of Rathke’s pouch in mice		Reviewed in (5)
	Bone morphogenic proteins	BMP2	Induces Isl1 expression Downregulation required for cell differentiation	Prolonged expression results in hyperplastic pituitary and lack of terminal differentiation		(65)
	Fibroblast growth factors	FGF 8, 10, 18	Expressed in the posterior pituitary Required for Lhx3 and Lhx4 expression and cell differentiation	In humans, mutations of FGF8 and its receptor are associated with Kallmann syndrome, resulting in isolated hypogonadotrophic hypogonadism		(66) (67)(68)
	Shh		Expressed in oral ectoderm and ventral diencephalon Induces Lhx3 expression	Antagonism in mouse oral ectoderm results in hypoplastic Rathke’s pouch		(69)
Lineage determination and cellular differentiation	Prop1		Transcriptional activator and suppressor depending on context Activates POU1F1 expression and switches developmental process from proliferation to differentiation	Mutations are most common cause of CPHD in humans		Reviewed in (6) and (70) (71)(72) (73)(74)
	POU1F1 (Pit1)		Expressed in cells committed to somatotroph, lactotroph and thyrotroph lineage Inhibits GATA2 and prevents gonadotroph cell fate	Mutations in humans associated with GH PRL, TSH deficiency and small anterior pituitary. Mutations rarely present in sporadic CPHD and more common in familial CPHD	Required for GH PRL, TSHβ expression	(73) (75)(76) (77)
	GATA2		Specifies gonadotroph and thyrotroph lineages Induces expression of Nr5a1	In mice, overexpression associated with gonadotroph and thyrotroph hypoplasia.	Expression persists in adult gland	(75)
	Nr5a1 (SF1)		Expressed throughout adrenal and reproductive axes Regulates expression of GnRHR, LH, FSH and αGSU. Expression necessary for gonadotroph differentiation	Mutations associated with 46XY sex reversal with adrenal failure, 46XY gonadal dysgenesis and 46XX ovarian insufficiency and premature ovarian failure in humans		(18) (78)
	Tbx19 (TPIT)		Activates POMC expression in association with PITX1	Mutations are commonest cause of isolated ACTH deficiency in humans	Antagonists to Nr5a1 can prevent gonadotroph cell fate	(79) (80)

Much of our understanding of the process of pituitary organogenesis comes from mouse studies (4) (5), but the phenotypes associated with human disorders often share aspects with mouse models of defective pituitary development. Various transcription factors that are involved are presented in Figure 1 and summarized in Table 1 (6), (7), (8) & (9).

Figure 1. Signaling molecules and transcription factors control pituitary development. Arrows represent regulation of expression in the direction indicated (See Table 1). The clinically used transcription factors are in blue.

Transcriptional Control of Pituitary Organogenesis: Lineage Determination and Cellular Differentiation

Significant advances have been made in recent years in elucidating the molecular mechanisms underlying the commitment and differentiation of Rathke’s pouch progenitors and the role of endocrine stem cells in the adenohypophysis (8) (10). Many factors are involved in pituitary differentiation, but a few later acting transcription factors have pituitary-specific effects and have become clinically relevant (11). In clinical terms, cells of the adenohypophysis can be divided into those of the Pit-1 lineage (somatotrophs, lactotrophs, somatomammotrophs, and thyrotrophs), those of t-PIT lineage (corticotrophs and melanotrophs), and those of SF-1 lineage (Gonadotrophs).

PIT-1 (POU1F1)

PIT-1 was first identified as a trans-activating factor of the growth hormone and prolactin genes. It is a member of the POU homeodomain family of transcription factors (5). Inactivating mutations produce recessive hypopituitarism characterized by a congenital lack of growth hormone, prolactin and TSH (12). Two naturally occurring recessive mouse mutants have Pit-1 defects; the Snell Dwarf (dw) mouse and the Jackson dwarf (dwJ). Homozygous recessive variants of these mice both exhibit post-natal, but not embryonic, anterior pituitary hypoplasia with GH, PRL and TSH deficiencies.

T-PIT

t-PIT (TBX19) is a member of the T-Box family of transcription factors. The T-Box represents the DNA binding domain of these factors. T-Pit is expressed in the developing pituitary and expression generally persists into the adult gland. T-Pit depletion in mice induces severe ACTH and glucocorticoid deficiencies in addition to adrenal hypoplasia and pigmentation defects. The phenotype of t-Pit ^-/- mice suggests that T-PIT promotes the development of corticotrophs but can also actively repress gonadotroph formation (13).

STEROIDOGENIC FACTOR (SF-1)

SF-1 is a nuclear receptor encoded by the gene Nr5a1 or steroidogenic factor 1 (SF-1). It is an orphan nuclear receptor involved as a transactivating factor in steroid biosynthesis. SF-1 is expressed throughout the adrenal and reproductive axes during development and postnatal life (14) (15). Mutations of SF-1 in humans are associated with 46XY sex reversal with adrenal failure, 46XY gonadal dysgenesis and 46XX ovarian insufficiency and premature ovarian failure (16) (17).

In some cases, transcription factor deficiency does not result in the complete absence of a cell type. SF-1 deficient mice do not spontaneously produce gonadotrophins but hyperstimulation with GnRH can induce hormone production suggesting that SF-1 is not essential for gonadotroph differentiation (18). Similarly, t-Pit expression is not essential for corticotroph development, but POMC synthesis is delayed if they are deficient (19). The failure to promote differentiation along a particular lineage can be permissive for other pathways and the idea that single transcription factors direct cell differentiation is overly simplistic. Nevertheless, these differentiation markers (colored blue in Figure 1) have proven to be extremely useful in diagnostic pituitary pathology, particularly in demonstrating the cell lineage of silent or non-expressing pituitary adenomas, and their expression can be detected in histological samples of pituitary tissue removed at trans-sphenoidal adenohypophysectomy by immunohistochemistry (Figure 2).

Figure 2. Pituitary transcription factor expression in the normal pituitary gland.

ANATOMY AND HISTOLOGY OF THE MATURE PITUITARY GLAND

Hypophysis Cerebri

The mature pituitary gland, or hypophysis cerebri, is a small, oval body weighing approximately 0.5g. The adenohypophysis of the pituitary is generally smaller in men than women and, within the female population, the gland in nulliparous women is generally smaller than that seen in multiparous women (20) (21). Indeed, during pregnancy, the gland may increase by approximately 30% due to a combination of lactotroph hyperplasia and vascular engorgement. The hypophysis is connected to the brain via the infundibulum, a tubular structure arising from the tuber cinereum and median eminence of the hypothalamus. The gland rests in the sella turcica (pituitary fossa) of the sphenoid bone and is covered superiorly by the diaphragma sellae (a thin film of dura), laterally by the walls of the cavernous sinus, and antero-inferiorly by the posterior wall of the sphenoid sinus; the latter used as the standard route for pituitary surgery or transsphenoidal adenectomy (Figure 3). Antero-superiorly, the pituitary lies in close proximity to the optic chiasm and bilateral, inferomedial compression of the chiasm by the expanding gland explains why space-occupying lesions of the pituitary commonly present with bitemporal hemianopia. Inferiorly, the adenohypophysis is separated from the floor of the sella turcica by a large, loculated venous sinus that communicates with the circular sinus.

Figure 3. High resolution 3D imaging reconstruction of the sella turcica in a volunteer (courtesy of Prof Mark Gurnell, Cambridge, UK). These detailed scans are proving invaluable in planning the surgical approach for transsphenoidal adenectomy, especially where vascular anomalies are expected.
A) Section across the roof of the sella with the optic nerves and chiasm (green) in situ revealing the intimate relationship between the chiasm and the pituitary gland (turquoise).
B) A similar level with the optic nerve and chiasm removed.
C) Detail of the sella region showing the relationship between the pituitary gland and stalk (turquoise), base of hypothalamus (orange), and internal carotid arteries (red).

The Hypothalamus

The structure of the hypothalamus and its complex connections are well known and have been described in detail elsewhere (22) (23) (24). The hypothalamic nuclei that give rise to the neurohypophysis arise from the preoptic, supraoptic, lateral, tuberal and mamillary regions (3) and hypothalamic regulatory neurons have their cell bodies in the supraoptic, paraventricular, infundibular and ventromedial nuclei of the hypothalamus. These hypothalamic nuclei have projections that terminate throughout the median eminence, infundibulum, and the posterior lobe (Figure 4). Neurons derived from hypothalamic nuclei produce peptidergic “releasing hormones” that are transported along axons and released into the portal system from where they are carried to the dense capillary plexus of the adenohypophysis to influence hormone synthesis and release. The parvocellular neurosecretory pathway of the tuberoinfundibular tract is proposed as the main route for the synthesis of releasing hormones and of transmission to portal blood. The major trophic factors originating in the hypothalamic nuclei include growth hormone releasing factor (GRF), corticotrophin releasing factor (CRF), thyrotropin releasing factor (TRF), gonadotrophin releasing hormone (GnRH) and other peptides such as somatostatin (SS). Other trophic and inhibitory factors are also released in this region including dopaminergic compounds.

Figure 4. Sagittal T1-weighted image of the pituitary fossa demonstrates normal pituitary anatomy. The T1-weighted study is particularly helpful in identifying the posterior pituitary bright spot (white arrow)- a focus of T1 signal hyperintensity posteriorly in the sella, which corresponds with the neurohypophysis. The intrinsic T1 signal shortening through the posterior pituitary is thought to be due to the presence of vasopressin. The zoomed image of the pituitary gland, consisting of the anterior (A) and posterior (P) lobes, sits within the sella turcica, a “saddle”-shaped depression in the sphenoid bone, with the tuberculum sellae anteriorly, and the dorsum sellae posteriorly. Superior to the pituitary gland is CSF within the hypophyseal and suprasellar cisterns. The pituitary stalk (S), also known as the infundibulum or infundibular stalk, extends through the suprasellar cistern, extending between the hypothalamus and the superior surface of the pituitary gland. The optic chiasm (C) can be seen in the suprasellar space more superiorly. Anteroinferior to the pituitary gland is the sphenoid sinus (SS). (B)- brainstem, (M)- mammillary bodies, (3V)- third ventricle.

In addition to nerve fibers, the long and short portal blood vessels (distributed between sinusoids in the median eminence, infundibulum, and the adenohypophysis) are also of major importance to this region. The adenohypophysis receives no major nerve supply and almost all of its blood comes from hypothalamic-hypophysial portal vessels. The predominant direction of blood flow is from the tufts of sinusoids in the median eminence and infundibulum via the portal vessels to sinusoids between glandular cells in the anterior lobe. This neurovascular arrangement is of vital importance to understand the mechanism by which the hypothalamus regulates the neuroendocrine function of the gland.

Neurosecretion is the physiological process involved in the production and release of posterior lobe oxytocin and vasopressin and their related proteins: including the precursor neurophysins. In terms of these magnocellular neurosecretory pathways, both the supraoptic and paraventricular nuclei are involved in the production both of vasopressin and oxytocin, however, vasopressin originates predominantly from the supraoptic nucleus and oxytocin originates predominantly from the paraventricular nucleus.

Infundibulum (Pituitary Stalk)

The infundibular stalk is a tubular, funnel-shaped structure divided into the anterior pars tuberalis and posterior pars infundibularis. The pars tuberalis is considered to be part of the adenohypophysis (see below) and contains a few scattered gonadotroph or corticotroph cells. Anteriorly and superficially, it surrounds the pars infundibularis (infundibular stem), which contains the unmyelinated axons of the magnocellular supraoptic and paraventricular neurons. The neurons contain large intra-axonal accumulations of oxytocin and vasopressin that may be seen as eosinophilic ovoid granular swellings along the trajectory of these axons in the infundibular stem. These ovoid swellings are known as “Herring bodies” named after Percy Theodore Herring who first described them (figure 4). It also contains the neural connections of the hypophysis that are continuous with the median eminence of the tuber cinereum.

The Adenohypophysis

The adenohypophysis comprises the vast majority of the pituitary gland by volume and is composed of three parts: the pars distalis, the pars intermedia and the pars tuberalis. The pars distalis forms the majority of the adenohypophysis, the pars intermedia is rudimentary in the human but represents the vestigial posterior limb of Rathke’s pouch and the pars tuberalis is an upward extension of the adenohypophysis that surrounds the lower regions of the hypophysial stalk. The anatomy of this region is shown in figure 5.

Figure 5. Coronal T2-weighted image demonstrates normal sellar and parasellar anatomy and delineates the local anatomical boundaries of the pituitary fossa. The pituitary gland (P) is sited within the sella turcica, with the sphenoid sinus (SS) demonstrated inferiorly. The pituitary stalk (S) is seen extending vertically through the suprasellar space in the midline. Superior to this is the optic chiasm (C). The lateral borders of the pituitary fossa are formed by the cavernous sinuses. On this coronal image, the T2 flow voids through the cavernous segments of the ICAs occupy the cavernous sinuses.

Pars Distalis

The pars distalis of the adenohypophysis is a highly vascular structure, consisting of epithelial cells of varying size and shape arranged in cords, irregular masses or follicles, separated by thin-walled vascular sinusoids and supported by a complex network of reticular tissue best appreciated in reticulin preparations (Figure 6). This region of the adenohypophysis contains several cell types, generally divided into acidophils and basophils (depending on their staining properties with mixed acidic and basic dyes such as Orange-G/aldehyde fuchsin, (figure 7). These cells release trophic hormones including growth hormone (GH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH) and luteinizing hormone (LH). The regulation and production of these trophic hormones is complex and is thought to be the result of a combination of the release of hypothalamic releasing factors into the median eminence, the regulation of portal blood flow by vascular systems, feedback by systemic hormones, autocrine interactions and complex paracrine interactions within the gland (25) (26)(27).

Figure 6. Section of adenohypophysis stained with reticulin showing nested acini of adenohypophysial cells.

Figure 7. Section of adenohypophysis stained with PAS/Orange G showing the distribution of acidophils (Orange) and basophils (Purple).

The cellular heterogeneity within acini may be demonstrated tinctorially in PAS-OG preparations (Figures 7, 8) which were widely used before the adoption of antibody-based stains (Figure 9). Corticotroph cells are generally strongly basophilic (PAS-positive), somatotroph and lactotroph cells mostly acidophilic (orangeophilic), whilst gonadotrophs and thyrotrophs may be basophilic or chromophobic (reacting with neither acid nor basic stains). There is no perfect match of hormone-expression and type or degree of chromophilia; some chromophobe cells may represent degranulated chromophil cells or precursor cells. In a normal adult adenohypophysis, approximately 10% of endocrine cells are basophils, 40% acidophils and 50% chromophobes.

Figure 8. The axial section of the adenohypophysis of the human pituitary gland at the level indicated by the dashed blue line in the diagram. Although there is a mixture of different hormone producing cells in most pituitary acini, the distribution of cells is not random: this is most pronounced in the ‘lateral wings’, which contain mostly somatotroph cells and the central ‘mucoid wedge’, which contains the majority of the corticotrophs. This is easily appreciated in periodic acid-Schiff / orange-G (PAS-OG) histochemistry, which stains somatotrophs yellow-orange (OG-positive) and corticotrophs purple (PAS-positive).

The PAS-OG stain is still a useful supplementary method in the differential diagnosis of some pituitary lesions (hyperplasia, corticotroph microadenoma, Crooke’s cell changes or adenoma) but these tinctorial stains have all largely been replaced by immunohistochemical methods (Figure 9). Although most acini contain a mixture of different hormone-producing cells, there is evidence of zonation (Figure 8). The lateral wings of the gland mostly contain somatotrophs and lactotrophs, whilst corticotrophs are concentrated in the median mucoid wedge, which at its anterior border (the rostral tip) harbors clusters of thyrotrophs. Gonadotroph (LH/FSH) cells are diffusely scattered throughout the gland.

Figure 9. Examples of immunohistochemistry. It can be seen that although growth hormone (GH) is generally enriched in the wings, the distribution is more diffuse. Corticotrophs (ACTH) are more obviously restricted to the central “mucoid wedge”. Compare this with tinctorial stains in Figures 7 & 8.

Cell Types of the Adenohypophysis

SOMATOTROPHS

Somatotrophs represent about 50% of cells in the adenohypophysis and are located predominantly, but not exclusively, in the lateral wings. They are readily identified by the expression of growth hormone. A subset of somatotrophs also express the common glycoprotein hormone alpha-subunit. They are the second cell type to form during fetal development and are detected by 8 weeks of gestation.

LACTOTROPHS

Lactotrophs express prolactin. The number of prolactin-secreting cells varies widely between the sexes and with parity. The Golgi apparatus is well developed in lactotrophs and contains immature, pleomorphic secretory granules. Granule extrusions are common in lactotrophs, not only at the basal cell surface, but also on the lateral cell borders; the latter is a distinctive feature of lactotrophs seen on electron microscopy and is known as “misplaced exocytosis”. Lactotrophs appear to arise from mammosomatotroph cells and pure lactotrophs appear late in gestation (24 weeks). The expansion and secretion of lactotrophs is controlled by exogenous hormones including estrogen, paracrine effects from adjacent adenohypophysial cells and inhibitory substances from the hypothalamus.

MAMMOSOMATOTROPHS

These bihormonal cells contain both growth hormone and prolactin. Mammosomatotrophs are the precursors of lactotrophs and, as in lactotrophs, misplaced exocytosis can be seen on electron microscopy (figure 10). These cells are thought to be the source of lactotroph increases during pregnancy (28)

Figure 10. Electron micrograph of a mammosomatotroph adenoma displaying misplaced exocytosis. This feature is seen as the extrusion of secretory granules into the extracellular spaces or from intercellular extensions of the basement membrane.

CORTICOTROPHS

Corticotrophs synthesis the molecule pro-opiomelanocrortin (POMC) which is cleaved post-transcriptionally to produce bioactive peptides including adrenocorticotrophic hormone (ACTH), melanotrophin (MSH) lipotrophic hormone (LPH) and endorphins (29). These cells comprise about 15-20% of the cell component of the adenohypophysis and the cells are concentrated in the central mucoid wedge. They are best identified histologically by using antibodies raised against ACTH. Corticotrophs are the first cell type to differentiate in the fetal pituitary gland at 6 weeks of gestation and ACTH can be detected at 7 weeks. Exposure to glucocorticoid access, either by exogenous corticosteroid administration or any cause of endogenous glucocorticoid hypersecretion, including ectopic secretion of ACTH, causes corticotrophs to undergo a distinctive, but reversible, morphological alteration known as Crooke hyaline change (30).

THYROTROPHS

These are the most infrequent cell type within the adenohypophysis and are detected by the expression of the glycoprotein hormone common alpha-subunit and the beta-subunit of TSH. This can be demonstrated by immunohistochemical double labelling for both subunits. Thyrotrophs are detectable at about 12 weeks of gestation.

GONADOTROPHS

Gonadotrophs produce both luteinizing hormone (LH) and follicle stimulating hormone (FSH) and both hormone specific beta-subunits and the common alpha-subunit can be detected in these cells. Gonadotrophs are scattered throughout the pars distalis and are the major constituent of the pars tuberalis. Gonadotrophs are intimately associated with lactotrophs and electron microscopy reveals gap junctions between them. The synthesis and release of the gonadotrophins is differentially regulated by the hormonal milieu, paracrine interactions and the pulsatile frequency of GnRH (31) (26,32).

There is growing evidence that the precise control of pituitary gland secretion also involves a contribution from cells with the capacity to demonstrate plasticity with self-renewal and it is likely that the pituitary contains a pool of stem cells (33)(34) (35) (36). It is possible that these stem cells, possibly expressing Nestin, Lhx3 and Sox2, may exhibit self-renewal properties and have the ability to differentiate into different hormone-producing cell types in response to differing physiological demands (36) (37) (38).

FOLLICULOSTELLATE CELLS

Folliculostellate (FS) cells are (in the adult human pituitary) an agranular (non-hormone-producing) parenchymal component of the pars anterior. It has been postulated that they represent a stem cell capable of trans-differentiation into endocrine cells (39), but whether this is true in humans is not certain. FS cells are small, chromophobe, with slender processes that extend between the endocrine cells. They form small follicles at the center of acini, comprised of apical tips of multiple FS cells. They may be visualized with antibodies raised against the S100 protein and GFAP, but their expression pattern is not always overlapping and may reflect different stages of maturation or function. In our hands, annexin-1 immunohistochemistry is a robust marker of FS cells (Figure 11). Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins. ANXA1-positive FS cells may modulate glucocorticoid feedback loops in the anterior gland (40) or act as antigen-presenting cells. FS cells are also postulated to play a role in paracrine regulation (26,41).

Figure 11. Non-endocrine cells of the anterior lobe include small folliculo-stellate (FS) cells with delicately branching processes that invest endocrine cells (left, annexin-1 staining) and (on the right) very rare cells that are postulated to represent adult pituitary stem cells (PSC, nestin staining).

Pars Tuberalis

The pars tuberalis is characterized by a large number of traversing vessels. Between these are cords or balls of undifferentiated cells admixed with acidophilic and basophilic cells. The secretion of adenohypophyseal hormones into the circulation appears to occur by exocytosis of the vesicular contents into the perivascular spaces of the sinusoids, the latter being lined with a fenestrated endothelium which facilitates diffusion into the bloodstream. The signal for secretion is the liberation of chemical releasing factors from neurons in the median eminence, and other hypothalamic centers, into the portal system of veins by which they are carried and distributed within the adenohypophysis.

Pars Intermedia

In contrast to rodents, the pars intermedia is rudimentary in adult humans (Figure 12). It represents a narrow zone between the adeno- and neurohypophysis often containing microscopic remnants of Rathke’s cleft. This zone may also contain scattered intensely PAS-positive corticotrophs, which may extend from the mucoid wedge of the adenohypophysis into the neurohypophysis. This so-called “basophil invasion” must not be confused with corticotroph microadenomas; it is believed to increase with age, and it has been suggested that these basophil cells are functionally distinct from classical ACTH-producing cells of the adenohypophysis and do not respond with hyaline degeneration (“Crooke’s cell change”) in the setting of systemic hypercortisolemia. Secretory cells of the pars intermedia have granules containing either alpha- or beta-endorphin; these cells have been shown to contain various peptide hormones including ACTH and alpha-MSH (melanocyte stimulating hormone).

Figure 12. The axial section of the human pituitary gland at the level of the vestigial intermediate lobe (approximately representing the boxed area in the diagram). Note the cluster of remnants of Rathke’s pouch / cleft (RC). The arcs indicate the posterior (center) and postero-medial (left and right) edges of the mucoid wedge (with scattered basophils) and pituitary wings (with scattered somatotrophs), respectively. The asterisk indicates basophil corticotrophs ‘spilling’ into the neurohypophysis (‘basophil invasion’, see figure 13).

Figure 13. ‘Basophil invasion’ of corticotrophs from the vestigial pars intermedia into the neurohypophysis (pars posterior of the pituitary gland). The dashed line represents the border between pars intermedia and pars posterior.

Neurohypophysis

The neurohypophysis does not contain neuroendocrine epithelial cells. Instead, it is composed of the axons arising from groups of hypothalamic neurons, most prominently those originating from magnocellular neurons of the supraoptic and paraventricular nuclei (Figure 14). Some of these axons are short and terminate in the median eminence and infundibular stem among the superior capillary beds of the venous portal circulation. Longer axons pass into the main neurohypophysis thereby forming the neurosecretory hypothalamo-hypophyseal tract with their terminals ending near the sinusoids of the posterior lobe. These neurosecretory granules mostly contain oxytocin or vasopressin and form axonal beads close to their termini. Whilst it is believed that normal astrocytes may populate (at least partially) the infundibulum, the axon terminals in the neurohypophysis are supported by so-called pituicytes, which are characterized by the expression of the TTF-1 transcription factor (absent in classic GFAP-positive astrocytes) (42) (43) (44). These cells show elongated processes often running in parallel with axons and demonstrate only patchy GFAP and S100 expression.

Figure 14. The cytoarchitecture of the neurophypophysis (right) is strikingly different from the adenohypophysis, which contains the nested (inset, left) collections of neuroendocrine cells (left). The neurohypophysis does not contain neurosecretory cell bodies; instead, it is composed of specialized glial cells (pituicytes) that – unique in the human brain – express the TTF-1 (thyroid transcription factor 1) protein in their nuclei (inset, top right). The neurohypophysis contains nerve endings of the oxytocin and vasopressin producing cells of the hypothalamus. Their large, distended nerve endings can be identified on routine stains as so-called Herring-bodies (arrow), named after Percy Theodore Herring (University of Edinburgh) who described them in 1908 as the ‘physiologically active principle’ of the posterior gland.

The Vessels of the Hypophysis & Hypothalamic-Hypophysial Portal System

The vessels of the hypophysis cerebri have been extensively described and reviewed (45) (46) (47) (48). The arteries of the hypophysis arise from the internal carotids via a single inferior and multiple superior hypophysial arteries. The arteries of the median eminence and infundibulum end in sprays of capillaries. In the median eminence, these form the external (mantle) plexus and the internal (deep) plexus that are both drained by the long portal vessels some of which terminate in the adenohypophysis. Short portal vessels run from the lower infundibulum to the pars anterior. Both types of portal vessel open into the vascular sinusoids lying between the secretory cords in the adenohypophysis, providing most of the blood; there is no direct arterial supply to this region (49) (50). The portal system is considered to carry hormone-secreting factors from the hypothalamus (46) (45) (49). Another notable feature of the rostral portion of the stalk are tortuous capillary loops surrounding a central capillary, termed gomitoli (51). These structures are composed of a central muscular artery surrounded by a spiral of capillaries. Although their function is unknown, the complexity of these gomitoli suggests that they may regulate the rate of blood flow to the adenohypophysis thereby regulating the flow of trophic factors.

The venous drainage of the neurohypophysis is by three possible routes: to the adenohypophysis via the long and short portal vessels, via the large inferior hypophyseal veins into the dural venous sinuses or to the hypothalamus via capillaries passing to the median eminence. The venous drainage carries hypophysial hormones from the gland to their target tissues and also facilitates feedback of secretions. In contrast, the venous drainage of the adenohypophysis appears restricted. It is possible that flow in the short postal vessels could be reversed explaining the postulated “short feedback loop”. These models of hypophysial blood flow are of importance to the mechanism of hormone secretion. It is not certain whether the median eminence represents the final common pathway for neural control of the adenohypophysis or the entire neurohypophysis may be involved; its capillary bed may selectively determine the destination of both hypothalamic and pituitary secretions, conveying some to the glandular pituitary, others to distant target organs and yet others to the brain (48).

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Graves’ Disease and the Manifestations of Thyrotoxicosis

ABSTRACT

Graves' disease (GD) is an autoimmune disorder characterized by hyperthyroidism and various systemic manifestations, including thyroid eye disease (TED) and, less commonly, dermopathy. This chapter provides an in-depth review of GD, covering its history, epidemiology, risk factors, and the molecular mechanisms underlying autoimmune hyperthyroidism. Emphasis is placed on emerging insights into the genetic, environmental, and immunological factors contributing to GD's multifactorial pathogenesis. The chapter also explores the pathogenic role of TSH receptor antibodies and their significance in diagnosis and treatment, alongside key clinical features of thyrotoxicosis.

INTRODUCTION

Graves' disease (GD) is an autoimmune form of hyperthyroidism characterized by multisystemic manifestations of thyrotoxicosis and, in some cases, extrathyroidal manifestations of thyroid autoimmunity including, frequently, thyroid eye disease (TED) and rarely pretibial dermopathy. There are therefore diverse phenotypic presentations of thyroid autoimmunity, and patients do not necessarily present with thyrotoxicosis as the main manifestation of their autoimmune phenomena. Autoimmune thyroid disease (AITD) typically includes two major categories of manifestations. Those specific to GD, and caused by the autoimmunity per se, including goiter, TED, and dermatopathy (TED and dermopathy are discussed in the chapter “Graves’ disease: Complications”). The second set of problems is caused by the excess thyroid hormone and its widespread disturbance in metabolism. These thyrotoxic manifestations do not differ from those induced by any other cause of excess of thyroid hormone. Alternative etiologies of thyrotoxicosis are described in other chapters.

HISTORY

The triad of goiter, tachycardia, and ophthalmopathy was independently described by Caleb Parry (1755-1822) and Karl A. Von Basedow (1799 - 1854), but it is the description by the Irish physician Robert James Graves (1796 - 1853) (Figure 1) (1) that is the source of the acronym in the English literature, Graves' disease (GD). However, in many countries, the term Morbus Basedow is widely used to describe autoimmune hyperthyroidism. The autoimmunity reflected in GD is related to antibodies directed against the TSH receptor (TSH-R) expressed at the basolateral membrane of thyroid follicular cells. Initially, serum factors that were thought to be a modified TSH molecule and produced exophthalmos in experimental animal models were given the eponym exophthalmos producing substance (2, 3). Later, Adams et al. identified a factor in human serum that could stimulate the release of thyroid hormone in guinea pigs and a human subject (Figure 2) (4, 5). Because of the time course of its action being longer than TSH, this material was named Long-Acting Thyroid Stimulator, or LATS. Subsequent studies better characterized this substance as an immune gamma globulin that acts on a thyroid antigen, and mimics the action of the natural thyroid stimulator, TSH. Nearly three decades later, the antigen to which this antibody was directed was identified as the TSH-R (6, 7). Over the past four decades, there has been improved understanding of the accurate identification, quantitation, and pathophysiologic importance of the thyroid receptor antibodies (8-10), as will be further detailed throughout this chapter.

Figure 1. Robert James Graves, Statue. Marble statue of Robert James Graves, former president (1843–1849) of the Royal College of Physicians of Ireland. The statue was commissioned by John Henry Foley who died before completing it. Graves Hall, the Royal College of Physicians of Ireland, No. 6, Kildare Street, Dublin, Ireland. Photographer: Osama Shukir Muhammed Amin FRCP(Glasgow). Copyrighted work available under Creative Commons Attribution only license CC BY 4.0.

Figure 2. Stimulation of thyroid hormone secretion by LATS-P. The subject's thyroid iodine was labeled by administration of I131, and serial observations were made on the appearance of I131-labeled hormone in blood (Y axis) over one month (X axis). An infusion of 280 ml control plasma had no effect, but 280 ml plasma from a patient with Graves' disease caused a marked stimulation of secretion of hormone from the thyroid. (Adapted from: D.D. Adams et al., J. Clin. Endocrinol. Metab., 39:826, 1974).

EPIDEMIIOLOGY

GD has an estimated incidence of 30 to 80 cases per 100.000 persons per year and occurs more frequently in the white population while it appears to be less frequent in the Asian and Sub-Saharan African populations (3.8 to 5, and 0.7 to 51 cases per 1000.000 persons per year, respectively) (11-13). Female (lifetime risk 3%, eight-folds greater than in men) and middle-aged individuals (30 to 50 years of age) are at higher risk. The manifestations of disease tend to be more pronounced in patients younger than 65 years, while older patients tend to have an abbreviated presentation, with mainly cardiac manifestations (14, 15). Around 50% of patients with GD have a family history of autoimmune thyroid disease (AITD). Aside from the infrequent occurrence of postnatal thyrotoxicosis due to maternal antibodies, the incidence of spontaneous GD in children before the age of ten is unusual, but the incidence climbs with each decade until about age 60 (16-20).

RISK FACTORS

GD is a consequence of autoimmunity against the TSH-R. The next question revolves around the trigger for this specific autoimmunity. Contemporary understanding is that a multi-factorial process allows thyroid self-reactivity to occur. While our immune system is designed to prevent self-reactivity, to some extent, exceptionally low levels of self-reactivity are normally present (21). Sex, environmental, immunologic, and genetic factors interact to augment this immune response to a degree that predisposes to clinically manifest autoimmunity (Figure 3). Several such factors have been identified with some certainty, and others have been suggested.

Figure 3. Risk factors for Graves’ disease. MHC: Major histocompatibility complex; HLA: Human leukocyte antigen; CTLA: Cytotoxic T-lymphocyte-associated protein; RNA: Ribonucleic acid. (Created with Biorender.com)

Sex

The clearest association with any autoimmune condition is female sex, which carries a 10-20-fold risk compared to men (22). Although, the mechanism behind the increased incidence of autoimmune conditions in female is not fully understood, several factors leading to greater autoreactivity towards the thyroid have been proposed. Polymorphisms in the estrogen receptor genes might be at least partially responsive for the increased susceptibility of female to developing GD (23). In addition, marked fluctuations in leptin and other hormonal secretion patterns throughout the different endocrine states during the female lifespan (Including puberty, pregnancy, and menopause) have been linked with changes in the regulation of the innate and adaptive immune systems, and disbalance between pro and anti-inflammatory states, which altogether increase the risk of developing autoimmune diseases (22, 24).

The post-partum period is associated with a higher risk of developing GD. This relates to the transition from a skewed T helper 2 (Th2) cell response that suppresses the immune response and antibody production (Including TSH-R antibodies) during pregnancy, to a post-partum state in which the proinflammatory Th1 cell response returns to normal (25). Furthermore, it has been shown that fetal cells from male infants can persist in the maternal circulation for up to 20 years (fetal micro chimerism). These cells have been found in frozen thyroid tissue specimens from patients with GD and thyroid nodules, and it is hypothesized that they could modulate AITD by induction of immune response or development of a graft-versus-host immune response to the mother (26).

Environmental Factors

Considerable information has accumulated about factors in the environment that can induce GD. Cigarette smoking increases the risk of GD and complicates its extra thyroidal manifestations through direct cell damage, downregulation of the innate immune system, and induction of epigenetic changes (27-31). Conversely, moderate alcohol use and obesity seem to decrease the risk of developing the disease (32), while physical activity seems neutral in that respect (33). Other factors include:

INFECTIONS AND MOLECULAR MIMICRY

A persistent theory on the etiology of autoimmune diseases is that exposure to a particular peptide epitope in an environmental antigen might lead to immune reactivity to an amino acid sequence identical to that present in a human antigen such as the TSH-R, thyroperoxidase (TPO), or thyroglobulin (Tg). Through this molecular mimicry, exposure to a virus or bacteria could produce heightened immune reactivity towards these autoantigens. This sequence is believed to play a role in rheumatic fever and glomerulonephritis. There is some evidence that proteins present in a common intestinal parasite, Yersinia enterocolitica, may induce antibody reactivity to the TSH-R (34-36). While a clear association has not been established, a higher proportion of patients with GD have been infected with this bacteria than people without the disease (34) and exposure to the bacteria can induce TSH-R antibodies (TRAb) (35).

There is also evidence that autoimmunity to the thyroid can be induced by infection with the human T-lymphotropic virus type 1 (HTLV-1), which causes lymphocytic leukemia (37). Whether this is due to molecular mimicry of the virus, viral damage to the thyroid, or stimulation through another mechanism such as cytokine secretion, remains uncertain. In addition, it has been suggested that Helicobacter pylori (H. pylori) antigens may be involved in the development of AITD, and one study reported that up to 85% of patients with autoimmune atrophic thyroiditis have H. pylori infections (38). While molecular mimicry remains a tantalizing explanation, the factual evidence for its role in the pathogenesis of GD is minimal.

THYROID INJURY AND ANTIGEN RELEASE

It is accepted that certain types of injury to the thyroid are followed by the development of thyroid autoimmunity, including GD. In fact, this is one of the few proven causes of GD. The release of thyroid antigens following destruction of thyroid tissue may add a significant stimulation to a latent low level of thyroid autoimmunity, causing the development of GD. Radiation to the thyroid has been associated with a subsequent higher incidence of positive thyroid antibody tests (39), and an increased risk of GD, Hashimoto's thyroiditis, and TED (40, 41). Around 1% of patients with autonomous thyroid nodules who undergo radioactive iodide treatment or ethanol injection can subsequently develop GD (42, 43), with a tenfold higher risk if anti-TPO antibodies are present (44). Whether viral injury, as in the case of HTLV-1 and SARS-CoV (45, 46), plays a similar role in human AITD is uncertain. However, Covid-19 disease caused by the SARS-CoV has been associated with higher risk of developing subacute thyroiditis, GD, and TED.

IMMUNE THERAPIES

Administration of cytokines such as INF-α, IL-2, and GM-CSF can augment AITD, or in some cases appear to induce it de novo (47-49). Alemtuzumab therapy, used for depletion of circulating lymphocytes in the therapy of multiple sclerosis is followed by development of GD in a third of such treated patient within 6 months of recovery from T cell depletion (50). This and other immunosuppressive treatments may deviate the immune system from a Th1 to a Th2 type of response, reducing the number of regulatory T cells or altering the balance between T helper 17 (Th17) cells and regulatory T cells, and leading to overexpression of pro-inflammatory cytokines IL-17, IL-22, IL-23R, and IL-32 that are commonly seen in patients with AIDT (51-55). In addition, treatment with INF-α for chronic hepatitis or INF-β for multiple sclerosis has also been suggested to cause AITD and destructive thyroiditis, especially in females (RR: 4.4) and people with pre-existing anti-TPO antibodies (RR: 3.9) (56); however, a definitive association has not been established (57). In addition, while immune check point inhibitors can commonly induce hypothyroidism and thyrotoxicosis due to destructive thyroiditis (58), in rare instances they can also induce GD (59, 60).

EXCESS IODINE

Excess iodide can induce hyperthyroidism in patients with multinodular goiter (61, 62), a phenomenon known as “Jod Basedow”. Presumably, autonomous nodules in the goiter are in a subclinical state and unable to produce an excess of thyroid hormones as their synthesis is limited by iodide supply; yet, when the iodine supply is high, the autonomous nodules produce an excess of hormone. Well studied epidemics of iodide-induced thyrotoxicosis occurred for example in Tasmania, Denmark, and India after the introduction of salt iodization and were clearly associated with multinodular goiters rather than typical GD (63-66). In addition, increased iodine intake can actually augment thyroid autoimmunity through other mechanisms such as heavy iodination of Tg which appears to increase immunogenicity in animal studies (67), or hypothetical induction of follicular thyroid cell injury with secondary liberation of thyroid related antigens (68, 69). On the other hand, the addition of 2 - 6 mg per day of iodide to the intake of most patients with GD causes a dramatic, but probably temporary, reduction in hormone release, a phenomenon referred to as Plummer effect (70-73). Overall, whether an excess of iodine can induce true GD and autoimmunity remains unknown.

STRESS

The incidence of GD increased in Denmark during World War II (74), and in Serbia during the civil war in the former Yugoslavia (75). In general, studies have shown mixed results; however, meta-analysis suggests physical trauma and psychologic stress are possible environmental triggers of GD (76). Some authors indicated that patients with GD had suffered on average more stressful episodes than control subjects, but other similar studies failed to show this relationship (77-80). Stress induces a variety of physiologic responses including anxiety, tachycardia, and restlessness, among others, which are not unlike symptoms of GD. Its role remains enigmatic in causation of GD to this date. Theoretically, stress might cause activation of the adrenal cortex or the sympathetic nervous system which might cause stimulation of thyroid secretion, as has been shown in experimental animals (81). Other specific stressors have been reported. Aggressive weight loss programs that involve using exogenous thyroid hormone have been reported to induce GD (82).

Immunologic Factors

THYMIC SELECTION OF LYMPHOCYTES

Lymphocytes develop from precursors present in the bone marrow that undergo progressive maturation and selection in the thymus. Lymphocytes which fail to recognize endogenous human leukocyte antigens (HLA) undergo negative selection, as do those which strongly react with endogenous epitopes presented by HLA molecules (83, 84). In this process, more than 95% of all lymphocytes undergo apoptosis. As with other human molecules, thyroid molecules like the sodium iodine symporter (NIS), TSH-R, TPO, and Tg-RNAs are presented as immunoreactive peptides in the human thymus as part of the lymphocyte selection process (85, 86). Pre-T lymphocytes are thus educated in the thymus to recognize thyroid-related epitopes, and to generate self-tolerance against them. Expression of these thyroid antigens in the fetal thymus is under control of the AIRE gene, and absence of this gene leads to polyglandular autoimmune syndrome type 1 whose hallmarks are adrenal insufficiency, hypoparathyroidism and muco-candidiasis. In Down’s syndrome, despite having 3 copies of the AIRE gene, expression of thyroid antigens in the thymus is reduced, and this is thought to cause the elevated incidence of autoimmunity in these patients (87). Clearly the thymic selection process is imperfect, in a developmental process designed to provide the maximum repertoire of lymphocytes, some lymphocytes which weakly recognize autologous thyroid antigens do persist in the circulation, and can be found in normal patients and those with AITD (Table 1) (85).

REGULATORY T CELL ABNORMALITIES

Every T cell-mediated response is tempered by the interaction of effector and regulatory cells (88). Thus, a disproportionately low number or diminished function of regulatory T cells is, in part, responsible for the development of autoimmunity (89, 90). Several studies have demonstrated abnormalities in T cell regulation in GD with conflicting results. Some groups have proposed that patients with GD have a lower proportion of regulatory T cells (91-95), while others report a normal or high number of T regulatory cells but with impaired immune suppressive function (91, 96-100). In addition, Marazuela et al. reported a high proportion of defective intrathyroidal regulatory T cells in patients with autoimmune thyroiditis (101). Gangi et al. found that administration of GM-CSF induced development of regulatory T cells capable of suppressing immunity to Tg in mice (102). Molteni et al. reported that CD8 T cells can induce anergy in TSH-R specific CD4 T cell clones to prevent thyroid related auto reactivity (103) and, ultimately, Vaidya et al. reported higher numbers of naïve activated T cells and lower memory T cells in patients with TED (104). Overall, while there remains some uncertainty, decreased T regulation due to low cell counts or impaired function is involved in the development of GD.

EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLASS II

Exposure of thyroid epithelial cells to interferon, presumably secreted from infiltrating lymphocytes or other immune cells, can lead to the expression of MHC class II molecules on the thyroid cell surface (105, 106). Expression of these molecules allows the thyroid epithelial cell to function as a weak antigen presenting cell (APC) (107, 108). Culture of human thyroid cells from patients with GD in vitro shows that the expression of MHC class II molecules expression disappears (109), as it does when the cells are transplanted into nude mice (110). This is a compelling argument indicating that MHC class II expression on GD thyroid cells is secondary rather than primary event.

Table 1. Possible Factors in the Immunological Etiology of Graves' Disease
- Persistence of some autoreactive T cells and B cells (failure of negative selection) - Genetic polymorphisms - Re-exposure to antigens secondary to thyroid cell damage - Reduced or dysfunctional regulatory T cells - Cross-reacting epitopes on environmental and thyroid antigens - Inappropriate HLA-DR expression - Mutated T or B cell clones - Activation of T cells by polyclonal stimuli - Stimulation of the thyroid by cytokines
*HLA:* Human leukocyte antigen; CTLA:* Cytotoxic T-lymphocyte-associated protein*

Table 1. Possible Factors in the Immunological Etiology of Graves' Disease

- Persistence of some autoreactive T cells and B cells (failure of negative selection)

- Genetic polymorphisms

- Re-exposure to antigens secondary to thyroid cell damage

- Reduced or dysfunctional regulatory T cells

- Cross-reacting epitopes on environmental and thyroid antigens

- Inappropriate HLA-DR expression

- Mutated T or B cell clones

- Activation of T cells by polyclonal stimuli

- Stimulation of the thyroid by cytokines

HLA: Human leukocyte antigen; CTLA: Cytotoxic T-lymphocyte-associated protein

Genetic Factors

The increased incidence of GD in certain families and in identical twins has for decades indicated a powerful genetic influence on disease development (111-113). Studies of large samples of pairs of twins suggest that the genetic factors account for 73-79% of the liability to have positive TSH-R antibodies and to develop GD, whereas environmental factors account presumably for the remainder (113, 114). Adjusting for covariates (age, TSH and others), the estimate for genetic influence on serum anti-TPO and anti TG antibodies was also found to be between 60 – 80%. This inheritance turns out to be polygenic. Rather than inheriting one gene which, in a dominant fashion, would induce GD, individuals inherit many different genetic polymorphisms which are conducive to the development of AITD (115-117).

HUMAN LEUKOCYTE ANTIGEN (HLA) MOLECULES

HLA molecules are expressed in different isotypes. There are between 50 and 100 different HLA-DR molecules, and a much smaller number of HLA-DQ and HLA-DP molecules, all coded on chromosome 6, in the human genome (118). Of these, the HLA-DR are most abundantly expressed and most important. These molecules exist as dimers on the surface of antigen presenting cells. In the initiation of an immune response, the antigen presenting cell displays a specific epitope complexed in an HLA-DR. Recognition of this bi-molecular complex by the T cell receptor leads to stimulation of T cells. The amino acid sequences of the HLA-DR molecule determine the shape and affinity to the antigen presenting cleft (119). Thus, certain HLA-DR molecules are more efficient to present certain epitopes during the lymphocyte selection and maturation process in the thymus (84). Therefore, the inheritance of certain HLA genes, and the matching of the HLA-DR molecules with the structure of the TSH-R epitopes, or other thyroid related epitopes, plays a significant role in determining the development of AITD.

The first genetic factor to be associated with GD was HLA-B8 (120), a class I major histocompatibility component (MHC). Subsequently, this relation was found to be more specifically significant with an MHC class II molecule, HLA-DR3 (117). Inheritance of this gene, expressed on the surface of antigen presenting cells, was found to confer up to 5.7-fold increased risk of developing GD (121-123). Additional data demonstrated that the HLA molecules DQA1*0501, DRB1*0301, DQB1*0202, DQB1*0603, DQB1*0609, DQB1*0302, DQB1*0303 were also closely associated with higher risk of developing GD (124-128). In contrast, inheritance of HLA-DR beta 1*07, DQB1*0201, DQB1*0502, and DQB1*0602 appear to be protective (128, 129).

CYTOTOXIC T LYMPHOCYTE ASSOCIATED PROTEIN (CTLA)

When an immune reaction begins, the “first signal” is the recognition by the T cell receptor of an HLA molecule. If only one signal occurs, the T cell tends to be turned off or “anergized.” In order for a progressive immune response to occur, there must be a "second signal" provided by one of several adhesion molecules which exist on the antigen presenting cells (APCs) and T cells, and which tend to augment the affinity of the interaction (130). Of these, one of the most important is "B7", which exists in two forms, B7.1 and B7.2, present on the surface of APCs. These molecules interact with their cognate receptors on the T cell, CD28 for B7.1 and CTLA-4 for B7.2. In many situations interaction between B7.1 and CD28 give a positive stimulus to growth of the T cell, whereas interaction of B7.1 with CTLA-4 provides a negative signal, suppressing autoimmunity (131). CTLA-4 exists as a gene with several isoforms, and the inheritance of some of them is associated with a higher incidence of GD (132-137), and increased production of anti-TPO and anti-Tg antibodies (138).

Interestingly, the HLA association suggests a relationship to GD specifically, since it has to do with the presentation of specific antigen epitopes, whereas the CTLA-4 polymorphism appears to be a general phenomenon, allowing one population group to have augmented lymphocyte proliferation, but is not specifically related to thyroid disease. These observations also fit with the concept that development of GD is mediated by a set of genes rather than one specific gene.

NON-CODING RNAs

The RNA transcripts that regulate genetic transcription and protein translation, but do not encode a specific protein themselves, are denominated non-coding RNAs (139). These regulators play a key role in immune activation, cellular proliferation, and cytokine production. Thus, their abnormal expression can halt the normal immune response, leading to autoimmunity. In the case of AITD, the understanding of non-coding RNAs associated with development of disease is still limited. Several transcripts have been proposed to play a role in the development of GD but their exact mechanism of action, and their utility for clinical care are unclear (140-142). Ongoing and future efforts aim to clarify the role of non-coding RNAs as well as their potential use as diagnostic biomarkers or therapeutic targets (143).

EXOSOMES

Exosomes are cell-secreted extracellular vesicles that function as biological carriers, and contain a series of molecules (i.e., cytokines, transduction factors, nucleic acids, proteins, and lipids) involved in processes of cellular communication such as antigen presentation and immune response activation. As in other autoimmune conditions, exosomes are presumed to play a role in AITD but research on their pathophysiologic role is still in the early stages (144, 145). Some authors have previously reported the presence of thyroid follicular cell derived exosomes containing TSH-R which are involved in the development of GD (146, 147). In addition, exosomes from patients with GD have been found to stimulate the production of proinflammatory cytokines such as IL-1, IL-6, and TNF-α (148). Furthermore, exosomes containing metalloproteinases and non-coding RNAs have been found in high proportions in patients with AITD and TED (149, 150).

OTHER FACTORS

Numerous other gene polymorphisms have been reported to be associated with GD. It is highly likely that these individual variants contribute a real but small increment in the risk of developing the disease. The initial genetic studies on the TSH-R showed mixed results on a potential association between specific polymorphisms and susceptibility to GD (151, 152). However, more recent studies have endorsed this association (153, 154). Certain genetic variants in TPO and Tg have also been associated with TED and AITD but their role in the development of disease is unclear (155-159). In addition, a vitamin D receptor polymorphisms have been associated with GD and other autoimmune conditions (160). Inheritance of specific genes coding for immunoglobulins may carry the same kind of risk. Additionally, several possible genes linked to GD or AITD have been found by linkage studies, including one recently described at a locus on chromosome 18q21 that is also associated with type 1 diabetes (161).

Other gene polymorphisms can lead to increased susceptibility to GD including CD40, CD25, and ZFAT (required for the correct functioning of B cells and other immune cells) (117, 162-164), CYP27B1 (involved in the activation of vitamin D) (165), IL-6 (166), IL-13 (167), IL-1 (168), IL-23R (169), TNF-α (170), protein tyrosine phosphatase-22 (PTPN22) (involved in inhibition of T cell activation) (171-174), Fc receptor-like protein 3 (FCRL3)(175-177), transforming growth factor beta1 (TGF-β1) (178), interferon inducible helicase 1 (IFIH-1)(179). Other proposed genes associated with AITD include FOXP3, TBX21, HLX, BTNL2, NOTCH4, and CXCR4 (158, 180).

In conclusion, there are several genes harboring polymorphisms that appear to augment the possibility of developing immunity to thyroid gland protein components. These genetic alterations are likely to interplay with epigenetic aspects of DNA methylation and histone modifications (a phenomenon referred to as epistasis), along with environmental interventions, leading to the overall individual susceptibility to GD.

PATHOGENESIS

GD and Hashimoto's thyroiditis are strongly associated and, in fact, overlapping syndromes. They share immunological abnormalities, histological thyroid changes, and genetic predisposition. Patients can switch from one category to the other, depending upon the stage of their illness. For example, an individual might first present with hypothyroidism, thyroid enlargement and positive anti-thyroperoxidase (TPO) antibodies, and thus qualify as having Hashimoto's thyroiditis. At a later stage, this individual might become hyperthyroid with positive TSH-R autoantibodies and fit in the category of GD. Conversely, a patient with hyperthyroidism might have progressive destruction of the thyroid, or develop blocking antibodies to the TSH-R, and become hypothyroid. The aspects explaining the autoimmunity and pathologic changes at the thyroid level are explained in this section. Additional thyroidal pathophysiologic changes are explained later in the chapter.

The common feature of AITD is the immune reactivity, both humoral and cell-mediated, to specific thyroid antigens such as the TSH-R, TPO, and Tg (21, 181). Antibodies also exist, among others, to megalin (the thyroid cell Tg receptor) (182), to the thyroidal iodide symporter (183), and to components of eye muscle and fibroblasts in patients with thyroid eye disease (184) (Table 2). Up to 90% of patients with GD have anti-TPO antibodies, and up to 50% have anti-Tg antibodies (185-189). Rarely patients have antibodies directed against T4 or T3 (187). Peripheral blood mononuclear cells (190), thyroid lymphocytes (191), and lymph node lymphocytes demonstrated cell-mediated immunity to TPO, Tg and TSH-R (192, 193), and to specific epitopes of these molecules (194-199). Anti-TPO antibodies are not known to play a role in GD, but they could cause a certain degree of cytotoxicity, as noted in couple in vitro studies using serum from AITD patients (200, 201). The functional consequence of having anti-Tg antibodies is uncertain, but they do not appear to cause thyroid cell destruction. Tg and anti-Tg antibody immune complexes are rarely deposited in the kidney basement membrane of the glomeruli and can, in extremely rare circumstances, produce nephritis (200, 202, 203).

Table 2. Antibodies in Graves' Disease
Antibody	Graves’ disease	Hashimoto’s thyroiditis	Healthy controls
TSH-receptor antibodies (TRab) (204-209) Thyroid stimulating antibodies (TSAb) TSH- blocking antibody (TBAb) Neutral antibodies	99%	10%	2%
Anti-thyroperoxidase antibodies (Anti-TPOAb) (204, 205)	80%	90-95%	10-15%
Anti-thyroglobulin antibodies (Anti-TgAb) (205, 210, 211)	30-60%	60-80%	8-11.5%

In addition, patients with AITD often develop other organ-specific antibodies, and have higher risk of developing associated conditions such as Addison's disease, premature ovarian failure, chronic hepatitis, celiac disease, primary biliary cirrhosis, pernicious anemia, type 1 diabetes, multiple sclerosis, myasthenia gravis, vitiligo, rheumatoid arthritis, systemic lupus erythematous, systemic sclerosis, idiopathic thrombocytopenic purpura, urticaria, alopecia, and angioedema (212-220). The most frequent extrathyroidal antibodies include antibodies directed to gastric parietal cells, found in 50% of patients with Hashimoto’s thyroiditis (221), to adrenal steroidogenic enzymes, ovarian steroidogenic enzymes, components of the pituitary gland (222), DNA (223), liver mitochondria GD(224), and to cardiolipin (225). Further evidence of ongoing inflammation in GD is the elevation of ICAM-1, IL-6 and IL-8 cytokines seen in hyperthyroid patients (217, 226).

TSH Receptor Antibodies

These antibodies can be classified into three major categories:

Thyroid stimulating antibodies (TSab) interact with the TSH-R and stimulate adenyl cyclase and the protein kinase A pathway, as well as the phospholipase C pathway, thereby triggering thyroid hormone synthesis and cell proliferation (227-229). Functionally, this is identical to the effects induced by TSH itself. When TSab reaches a certain level of function, they cause an increase in thyroid hormone synthesis and secretion, as well as growth of the gland, and cause hyperthyroidism.
Thyroid blocking antibodies (TBAb) interact with the TSH-R by binding to different epitopes on the receptor, and they can block the binding of TSH to the receptor without stimulating function themselves (230-233).
Thyroid neutral antibodies (TNab) bind mostly to the hinge region of the TSH-R. While these antibodies neither stimulate nor inhibit its function, they appear to be involved in signaling activity leading to apoptosis of the thyroid follicular cells (234-236).

All these antibodies are commonly recognized by assays which detect their ability to interfere with the binding of TSH to the receptor and are identified as thyrotropin receptor antibody (TRAb), or Thyrotropin binding inhibiting immunoglobulins (TBII) (231). Probably all patients with GD have a mixture of all these three subtypes of antibodies.

TRAb/TBII can be measured with enzyme linked immunosorbent assays (ELISA) that measure the ability of the patient’s serum antibodies to inhibit binding of labeled TSH-R ligands (human monoclonal thyroid antibodies) to the TSH-R (209). In addition, TSab can be identified by a bioassay which quantifies the ability of the antibodies to stimulate the adenyl cyclase function of the TSH-R. Either thyroid cells or thyroid cell membranes can be used, and the cyclic AMP produced by this stimulation is quantitated by a luciferase-based assay (209, 227). A cyclic AMP responsive luciferase construct introduced into Chinese hamster ovary (CHO) cells, allows a sensitive luminescent assay for thyroid stimulating antibodies with the capability of high throughput suitable for use in general laboratories (228).

The TSH-R is initially formed as a single polypeptide inserted into the thyroid cell membrane. After processing, it becomes a two-peptide structure, one extracellular and one transmembrane, with the chains held together by disulfide bonds. Subsequently, reduction of the disulfide bonds by a disulfide isomerase may separate the two molecules and lead to shedding of the “alpha” subunit, which can be augmented by TSH stimulation of thyroid cells (237). The amino-terminal ectodomain of the human TSH-R has been expressed on the surface of CHO cells as a glycosylphosphatidylinositol-anchored molecule. This material can be released from the cells and is biologically active by binding immunoglobulins from serum of patients with GD, and displaying saturable binding of TSH (8), indicating that all of the "immunologic information" related to production of antibodies resides in the extracellular portion of TSH-R.

The initial bioassay developed by Adams et al. (5), and then by McKenzie (238), could quantitate TSAb (or LATS as it was then known) in up to 60% of patients with active GD. Newer assays measuring cyclic-AMP formation can detect TSab in over 99% of patients with GD and thyrotoxicosis (239), with a sensitivity of 97% to identify patients with GD, and 93% for patients with TED (240). The presence of TSab is characteristic of active GD, and if the thyroid can respond (e.g., has not been ablated with RAI), hyperthyroidism ensues. If untreated, the thyroid may be destroyed by the ongoing immune process, or blocking antibodies can develop, and the patient may become hypothyroid. In addition, the coexistence of TSab and TBab can cause a pull-push effect leading to shifting between hypo- and hyperthyroidism (241, 242).

During antithyroid therapy, TSab tends to decline. If their titer becomes undetectable there is a high likelihood of disease remission. However, if present in significant concentration, remission is very unlikely, explaining the failure of safely discontinuing antithyroid drug therapy (ATD) in more than 50% of patients after 12-18 months of treatment (243, 244). Similarly, TSab tends to decline after thyroidectomy (245). After radioactive iodide therapy, TSab titer increases for up to a year, probably because of the release of thyroidal antigens (246-248). Antibody levels gradually decrease during the subsequent years, reaching pretherapy values around the third year. During ATD, it is speculated that some immune modulation might occur, and the predominant TSab are replaced by TNab or Tbab.

The specific epitopes to which the TSab bind are in the amino terminal portion of the extracellular domain of the TSR-R and have been better characterized (249, 250). This has led to therapeutic efforts to induce tolerance to these epitopes (251, 252). The blocking antibodies tend to bind to sequences at the carboxy terminal portion of the receptor, closer to the plasma membrane, probably preventing the activation of the receptor through inhibition of signal-transmission required conformational changes (253). Details of these actions are revealed by cryo-electron microscopy analysis of the full-length TSH-R structure complexed with the inhibitory antibody K1-70™. This approach revealed key interactions between the receptor's transmembrane and extracellular domains, suggesting that K1-70™ inhibits TSHR by binding without activating it, while the stimulatory autoantibody M22™ likely induces activation through conformational changes in the hinge region (254).

The Role of Cellular Immunity

Lymphocytes of patients with GD are reactive to the TSH-R (Figure 4), TPO, and synthetic TPO-derived peptides (190, 199, 200, 255). The interaction between antigen presenting cells (APCs) and T cells triggers a multimodal cellular and humoral response. T helper 1 (Th1) cell derived cytokines such as interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL) 1β, IL-6, IL-9, IL-10, and IL-11 play a dominant role in the early phase of AITD (256). Later, there is a predominant T helper 2 (Th2) cell response with its derived IL-4, IL-10, IL-17, and IL-23 (257-260). TSH-R peptides that have aspartic or glutamic acid in the fourth position of their binding motif are particularly susceptible at inducing immune responses in GD and animal T cells (190, 261). Immunoreactivity towards the TSH-R has been reported in samples of orbital fat and preadipocytes, and differentiation of preadipocytes into adipocytes with high TSH-R reactivity has been induced by TSH stimulation (262-264). In addition, immunity to the TSH-R plays a direct role in the development of TED, through the secretion of proinflammatory cytokines by T cells.

Figure 4. The role of cellular immunity. Schematic representation of TSH-R endocytosis by antigen presenting cells (APC), subsequent proteolysis and presentation to T cells, and stimulation of thyroid stimulating antibody production by B cells. (Created with Biorender.com).

PATHOPHYSIOLOGY

Thyroid Function

In GD, the thyroid gland is functioning at an accelerated rate. Serum TSab stimulate the TSH-R, as evidenced by the higher activity of the cell membrane adenyl cyclase activity in the thyroid tissue of patients with GD, compared to normal thyroids (265). The plasma iodine clearance, a reflection of the thyroid iodine uptake, is increased from the normal rate of 10-20 ml/min to 40-2000 ml/minute. For this reason, the percentage of a tracer dose of radioactive iodine (RAI, I¹²³) found in the thyroid gland at 12 hours is elevated and distributed in a homogeneous fashion (266). Thyroid hormones, Tg, and iodotyrosine - normally secreted in minute amounts - are released into the blood rapidly and at increased rates (73, 267, 268). Furthermore, the rate of turnover of plasma thyroid hormones is also increased. Accelerated degradation is probably secondary to hypermetabolism and is not a primary event (269), although it has been reported that accelerated T4 turnover can persist after treatment of thyrotoxicosis (270). Finally, after anti-thyroid drugs (ATD) the thyroid hormones tend to fall and that response, along with the required dose of ATD drug required to achieve that response, can be used as predictors of GD remission (271).

An interesting historical observation regarding thyroid function during GD is that the uptake of RAI by the thyroid is not suppressed by the administration of exogenous T4 or T3 (272, 273), even if large amounts of hormone were given (old studies performed before the autoimmune basis for GD was fully understood). Non-suppressibility is caused by stimulation of the thyroid by TSab, and independence of feedback control via TSH.

Iodine Effect on the Thyrotoxic Gland

Iodine affects the metabolism of the diffusely hyperplastic thyrotoxic gland in a way radically different from its action on the normal gland. Years ago, Plummer demonstrated that GD can be temporarily or permanently controlled by the administration of iodide (70). Administration of large doses of iodine to laboratory animals causes a temporary inhibition of iodine organification, the Wolff-Chaikoff effect (274). The same phenomenon occurs in humans, and thyrotoxic patients are especially sensitive to this effect. The thyroid uptake of I¹³¹ is acutely depressed in thyrotoxic patients by administration of 2 mg potassium iodine, whereas more than 5 mg is needed to depress uptake in normal subjects. Concentrations of serum iodine above 5 µg/dl block iodine uptake and binding in the thyrotoxic gland (70, 71, 73, 272, 275). The Wolff-Chaikoff effect is transient. With continuous iodine administration, I¹³¹ uptake and binding recommences. The adaptation to excess iodine in animals involves a reduction of iodine transport into the thyroid which lowers intrathyroidal iodine content and escape from the Wolff-Chaikoff block. This adaptation occurs independently of TSH action.

The biochemical mechanism of the Wolff-Chaikoff effect has not been fully elucidated, but it is partially mediated by the downregulation of the sodium-iodide symporter (NIS) (276). Iodine does not prevent TSH or TSab from binding to the TSH-R but inhibits both the TSH-stimulated adenyl cyclase production of cAMP and its derived actions. In addition, iodine also causes a marked reduction in the release of previously formed hormones from the thyrotoxic gland but does not completely prevent further hormone synthesis. This phenomenon has been repeatedly observed and helps explain the beneficial therapeutic effect of iodide in GD (73). Ochi et al. demonstrated that chronic administration of iodine in GD blocks the stimulating effect on hormone release from both TSH and TSab (277). The block of hormone release that occurs in the thyroid of GD can be observed, although not uniformly, in the normal gland and in the normal gland made hyperactive by repeated administration of exogenous TSH. While early laboratory studies on the effect of iodine on the thyroid gland suggested that methimazole can inhibit somewhat the Wolff-Chaikoff effect (278-280), more recent clinical data indicates their ultimate impact on systemic thyroid hormone levels to be additive (281, 282). Clinical practice therefore favors this combination for treatment of severe cases of hyperthyroidism (278-280).

Extrathyroidal Processes

The pathogenesis of extrathyroidal complications of GD such as TED and dermopathy is described in detail in another chapter. Recent evidence demonstrates that stimulation of the TSab on the TSH-R in the orbital fibroblasts can lead to a crosstalk of this receptor with the insulin growth factor 1 receptor (IGF-1R) which stimulates hyaluronan accumulation in the orbital muscles and transformation of orbital fibroblasts into adipocytes (283, 284). This mechanism is believed to play a key role in the pathogenesis of TED and is the basis for emergent therapeutic alternatives in patients with TED and, maybe, dermopathy. Antibodies binding to and directly stimulating the IGF-1R have also been proposed to play a role in this process. However, IGF-1R antibodies exist in sera from about 10% of normal subjects, and in a similar percentage of GD patients, and are stable over time, strongly suggesting they have no unique role in GD (285).

PATHOLOGY

The ophthalmic and dermatologic changes seen in TED and dermopathy are described in the chapter on the Complications of GD. The thyroid gland changes seen in GD and changes in extrathyroidal organs that can be seen with any case of thyrotoxicosis are described below.

Thyroid Gland

The essential lesion of GD is parenchymatous hypertrophy and hyperplasia (Figure 5). The central features are increased height of the epithelium from cuboidal to columnar, and varying sizes and shapes of the follicles with reduced colloid content (286, 287). Papillary infoldings, cytologic evidence of increased activity, hypertrophy of the Golgi apparatus, increased number of mitochondria, and increased vacuolization of colloid are also seen. In addition, between the follicles, there is a large array of capillaries, together with a characteristic lymphocyte and plasma cell infiltrate. This infiltrate may be mild and diffuse throughout the gland, but more typically occurs as aggregates of mononuclear cells and even lymphoid germinal centers, referred to as focal thyroiditis. Occasionally the histologic pattern completely overlaps that of Hashimoto's thyroiditis. All pathological changes tend to regress when euthyroidism is achieved.

Figure 5. Histology of a thyroid gland of a patient with Graves’ disease. Columnar epithelium, vacuolization of colloid, and significant lymphocytic infiltrate are present in the setting of diffuse parenchymal hypertrophy.

Extrathyroidal Changes

MUSCLE

Patients with Graves’s disease can have diffuse degenerative atrophy, fatty infiltration, loss of striation, vacuolization, and proliferation or degeneration of nuclei in striated muscles (288-290). Skeletal muscles can also experience interstitial myositis characterized by plasma cells, tissue macrophages, and atrophy of fibers (291). This partly explains the sense of persistent weakness that patients describe after the normalization of thyroid hormone levels. While cardiac and smooth muscles tend to be spared in most patients, myocardial degenerative lesions have been reported in thyrotoxicosis, with foci of cell necrosis, mononuclear infiltrates, and mucopolysaccharide deposits similar to those described in extraocular and skeletal muscles (290).

The extraocular muscle changes are specific for TED, whereas the remainder of the abnormalities may reflect the action of excess hormone.

PITUITARY

The anterior pituitary demonstrates a dramatic decrease in identifiable thyrotropin containing cells, based on data from patients who died from thyroid storm (292, 293). This is entirely reversed in patients who achieve euthyroidism after treatment.

LIVER

Initial studies based on autopsies of patients with GD suggested focal and even diffuse liver cell necrosis, atrophy, and cirrhosis (269). In more contemporary series of liver biopsy specimens obtained from thyrotoxic persons, the deviations from normal were minimal (294, 295). Moderate decrease in glycogen content and increase in fat and round cell infiltrates were noted. The differences among these studies could be explained by a lesser severity and duration of the disease in more recent series with patients diagnosed earlier and treated effectively, as the disease has now an extremely low mortality rate (296). Recent studies have documented that thyroid hormone analogs specific for the thyroid hormone receptor b such as resmetirom may be a novel therapeutic approach for the therapy of metabolic dysfunction associated steatotic liver disease (MASLD) (297).

BONE

Prolonged hyperthyroidism is known to produce the histologic picture of osteoporosis (298), but osteitis fibrosa also occurs (299). Histomorphometry studies show unmistakable evidence of excess bone formation and resorption.

NATURAL COURSE OF GD AND CLINICAL MANIFESTATIONS OF THYROTOXICOSIS

In the classic presentation, the most common onset is the simultaneous and gradual development, over a period of weeks or months, of thyrotoxicosis-related signs and symptoms, goiter, and exophthalmos. However, GD displays an array of possible clinical patterns. It is possible for GD to develop in a patient with preceding nontoxic multinodular goiter, toxic multinodular goiter (Marine-Lenhart syndrome), to experience thyrotoxicosis without TED, or to have TED preceding the development of thyrotoxicosis. Due to the coexistence of TSab and TBAb some patients can first develop hypothyroidism and later thyrotoxicosis, or vice versa (232). In addition, during pregnancy GD tends to be partially suppressed, only to have a rapid recurrence in the post-partum period (300). Sometimes, human chorionic gonadotrophin (hCG) induced thyrotoxicosis, seen in the first part of pregnancy, can also coexist with GD and mask its presentation (301).

Before the general availability of current therapies, hyperthyroidism tended to evolve through periods of exacerbation and remission. In the mild forms, the active disease was self-limited to one year or more, and the patients returned spontaneously to a euthyroid state. In untreated moderate to severe forms, mortality could be observed in 11% of patients (302, 303). Mortality was most frequently attributed to cardiovascular complications (such as myocardial infarction, arrhythmia, or heart failure), or infections, and occurred within the first 4 years after diagnosis. Fortunately, death due to hyperthyroidism is now rare with an excess mortality of 1.2% compared to controls (296). Nowadays, after a period of anti-thyroid drug therapy (ATD) therapy there is re-establishment of normal thyroid homeostasis and over time a sizable proportion of patients can achieve disease remission, allowing them to discontinue ATD and remain euthyroid off therapy.

The most common presenting symptoms are weight loss, weakness, dyspnea, palpitations, increased thirst or appetite, diarrhea, irritability, profuse diaphoresis, heat intolerance and increased tolerance to cold, or tremor. Occasionally, exophthalmos or diplopia is the index symptom, but goiter may antedate all other manifestations. The nutritional state varies greatly. In the past, patients were severely emaciated, but these days, on average, the weight loss is 5 - 20 lbs. (2.3 – 9 kg). Facial expressions of flushing, fright, or extreme anxiousness are common. Notably, in elderly patients the disease can manifest as apathetic thyrotoxicosis, in which there is absence of hyperkinetic neuromuscular symptoms, and predominance of cardiac (e.g. arrhythmia), psychiatric (e.g. apathy, depression) and nutritional (e.g.., weight loss) symptoms (304).

In patients with GD, the ocular changes of TED, lymphoid hyperplasia, localized abnormalities of skin and connective tissue (e.g., dermopathy and acropachy) and the goiter itself are direct results of the autoimmune processes of GD. The remainder of the changes are entirely attributable to an excess of thyroid hormone.

The natural course and manifestations of thyroid storm, TED, and dermopathy are primarily discussed in the chapter on Complications of GD. The clinical manifestations of thyrotoxicosis are described below.

Thyroid

The diffuse toxic goiter is usually more or less symmetric. The size is related, but not closely, to the severity of the disease. It varies from the barely palpable normal (10- 15 g) to a three-to-six times enlargement (45-100 g) or, rarely, even more. The gland might not be palpable in 1% of cases, either because the thyroid is smaller than usual at baseline or because it is beneath the manubrium. The thyroid may be smooth, lobulated, or rarely nodular. In thyrotoxicosis associated with nodular goiters, the hyperfunctioning tissue may reside between the nodules (305). Usually, the consistency is firm but elastic, or very firm if iodide has been given. The borders are easily demarcated by palpation. Thrills and bruits, usually denote the hypervascularity associated with increased function. Bruits may be continuous or systolic and are usually audible over the entire thyroid. Local pressure symptoms, including dysphagia and the sensation of a lump in the neck, are produced by the enlarged goiter. Vocal cord palsy is only rarely seen in GD (306). In addition, the supraclavicular lymph nodes could become enlarged and, rarely, tender (307). Most thyroid related manifestations tend to regress or disappear with restoration of euthyroidism.

Skin

Cutaneous manifestations are nearly always present when vasomotor overactivity is significant. Heat intolerance and profuse diaphoresis occur under circumstances that would provoke no response in normal people. On palpation, hands are usually erythematous, hot, and moist (hyperhidrosis). There may be continuous erythema of the face and neck, with superimposed transient blushing after palpation of the thyroid. Occasionally diffuse pruritus or urticaria occur (308). Patchy vitiligo is an associated manifestation of autoimmunity directed toward melanocytes that can be found in 7% of patients with GD.

Fingernails can experience onycholysis characterized by ragged appearance, thinning and posterior erosion of the hyponychium. The free margin of the nail leaves the nail bed, producing a concave or wavy margin at the line of contact. Temporary thinning of the hair is common, but alopecia is rare. Hair loss can occur due to marked changes in metabolism throughout the course of the disease.

Myxedema can be seen in 0.5-4.3% of patients with GD, and 13% of patients with TED (309, 310). The clinical characteristics of thyroid dermopathy are described in the chapter on Complications of GD.

Nervous System

Neural and mental findings are diverse. Nervousness, irritability, anxiety, and restlessness are common (Table 3) (311). The behavioral reactions to all sorts of stimuli are typically exaggerated. When asked to sit up, the patient might jump into an upright position. They may simply wish to cooperate but appear to overdo it. Often emotional instability is combined with this pattern, to the point of a notable change in personality. In some patients, the emotional pattern is that of mania or euphoria. In others, hyperactivity produces a state of exhaustion, and profound fatigue or asthenia. The mind is often highly active, and the patient is troubled with insomnia. Rarely, patients develop visual or auditory hallucinations or a frank psychosis. The latter is probably the result on an exacerbation of a baseline psychiatric condition rather than a de novo process (312). Furthermore, impairment of intellectual function has been found in patients with untreated hyperthyroidism. A recent study has documented that in patients over 65 years, a low TSH level from either endogenous or exogenous thyrotoxicosis is associated with higher risk of incident cognitive disorders (313).

A fine, rapid tremor of the outstretched fingers is classically found, and a generalized tremulousness, also involving the tongue, may be evident. The speed of muscle contraction and relaxation is increased, tendon reflexes tend to be brisk, and the reflex relaxation time is shortened (314). The tremor of Parkinsonism is intensified during thyrotoxicosis, and manifestations of cerebellar disease or pyramidal tract lesions can be seen (315, 316). Muscle fibrillations are not a usual part of the syndrome, but they may occur in chronic thyrotoxic myopathy. Polyneuropathy has also been reported (317). Rarely, patients manifest disorientation, aphasia, grimacing, choreoathetoid movements, symptoms suggestive of encephalitis, episodes of hemiparesis or bulbar paralysis (318). These symptoms clear up completely after restoration of an euthyroid state.

Other possible, although rare, severe neurologic manifestation consist of the new onset, exacerbation, or refractoriness of seizure disorders (319). Electroencephalography reveals increased fast wave activity, and occasionally bursts of tall spike waves. In addition, several reports describe a severe, steroid-responsive encephalopathy in some patients with GD (320).

Table 3. Neurologic Manifestations in Graves' Disease
*Common:* · Nervousness · Irritability · Restlessness · Anxiety · Emotional lability including hypomania or euphoria, and fatigue or asthenia). · Insomnia · Tremors · Brisk tendon reflexes with short relaxation time *Uncommon:* · Visual or auditory hallucinations · Psychosis · Impairment of intellectual function · Disorientation · Aphasia · Choreoathetoid movements · Hemiparesis or bulbar paralysis · Seizures · Encephalopathy · Neuropathy

Table 3. Neurologic Manifestations in Graves' Disease

Common:

· Nervousness

· Irritability

· Restlessness

· Anxiety

· Emotional lability including hypomania or euphoria, and fatigue or asthenia).

· Insomnia

· Tremors

· Brisk tendon reflexes with short relaxation time

Uncommon:

· Visual or auditory hallucinations

· Psychosis

· Impairment of intellectual function

· Disorientation

· Aphasia

· Choreoathetoid movements

· Hemiparesis or bulbar paralysis

· Seizures

· Encephalopathy

· Neuropathy

Muscular System

The muscular symptoms vary from mild myasthenia to profound muscular weakness and atrophy, especially of the proximal muscle groups. Wasting of the temporals and interossei used to be noted in a considerable number of patients while a few had generalized muscle wasting in the decades prior to current diagnostic and therapeutic abilities. Occasionally the myopathy may shade into the picture of polymyositis. Muscle cell necrosis and lymphocyte infiltration may be visible histologically, but usually are not found even when the symptoms of weakness are severe (321, 322). The electromyogram is normal in most instances but may occasionally resemble that of muscular dystrophy (323).

Work efficiency, measured in terms of the calories of heat produced while performing a given amount of work, has been reported to be either normal or decreased (324, 325). The muscles have decreased ability to incorporate creatine from the blood (326, 327). Creatinine excretion is initially increased by the general catabolism of hyperthyroidism, but as muscle mass diminishes, creatinine excretion in the urine decreases.

Myasthenia gravis may simulate thyrotoxicosis, and vice versa (328). The close relationship between these two diseases is apparent in the observation that thyrotoxicosis occurs in 3% of patients with myasthenia gravis. The pathogenic anti-acetylcholine receptor antibodies that occur in myasthenia gravis are clearly comparable to the TSH-R antibodies found in GD.

Periodic paralysis is precipitated and worsened by thyrotoxicosis (329). It has been more commonly reported in Southeast Asian males who have a higher frequency of certain genetic susceptibilities associated with higher risk of thyrotoxic periodic paralysis (330-333). Paralysis is usually associated with hypokalemia. Inactivating mutations in the inwardly rectifying potassium channel 2.6 encoded by the KCNJ18 gene (in about 30%) have, among other candidates, been associated with susceptibility for developing thyrotoxic periodic paralysis (334, 335). Beta adrenergic stimulation mediated by thyrotoxicosis augments sodium-potassium ATPase activity in the skeletal muscle leading to increased potassium uptake into the cells (336). The episodes of paralysis tend to be infrequent and sporadic, but most commonly occur after a meal, following exercise, or during sleep, and can be induced by administration of glucose and insulin. Episodes last from minutes to hours, usually involving peripheral muscles, but can cause paralysis of the diaphragm and affect the heart. Serious episodes can be associated with extensive muscle cell damage and necrosis, and electrocardiographic (EKG) abnormalities associated with hypokalemia, such as ST and T wave changes, premature ventricular complexes (PVCs), first degree heart block, prolonged QT intervals, and even ventricular fibrillation (337).

Pulmonary System

Except for dyspnea on exertion, symptoms deriving from the lungs are not prominent. Nevertheless, pulmonary function tests can show some reduction in vital capacity, expiratory reserve volume, pulmonary compliance, and airway resistance (338, 339). Minute volume response to exercise is excessive for the amount of oxygen consumed (340).

Cardiovascular System

The first and most common manifestations deriving from the cardiovascular system are palpitations and tachycardia (341, 342). The pulse on palpation is rapid and bounding. When present, it can be effectively controlled with beta- adrenergic blockers (343, 344). PVCs, paroxysmal atrial tachycardia, atrial fibrillation, and shorter P wave duration occur in 6-12% of patients (345-347). When present, atrial fibrillation should be treated with anticoagulation to prevent risk of thromboembolic complications. The systolic blood pressure is frequently elevated. The diastolic blood pressure is characteristically decreased, making the pulse pressure elevated to between 50-80 mm Hg (348). A systolic murmur can sometimes be heard over the precordium due to development of mitral valve prolapse following papillary muscle dysfunction during thyrotoxicosis (349). However, mitral valve insufficiency is usually not clinically relevant and can revert to normal with treatment of thyrotoxicosis.

In thyrotoxicosis, the heart rate, stroke volume, left ventricular mass index, and cardiac output are all increased. The pre-ejection period is shortened, and the left ventricular ejection time remains relatively normal. The interval from initiation of the QRS complex to arrival of the arterial pulse in the brachial artery is reduced (350). Circulation time is decreased. There is dilatation of superficial capillaries and decline in systemic vascular resistance (351). Coronary blood flow and myocardial oxygen consumption in each stroke are increased. Circulating plasma volume is increased (352). Long-term mild excess of thyroid hormone causes impaired cardiac reserve and exercise capacity (353). Cardiac enlargement and heart failure may occur with or without prior heart disease (354). These effects tend to normalize when euthyroidism is restored (355).

Patients with coronary artery disease often develop angina during thyrotoxicosis. However, angina could also develop de novo in patients with normal coronary arteries, especially young females. This condition has been ascribed to an imbalance between increased cardiac work and blood supply, even with a patent vessel (356). Severe coronary vasospasm has been observed during angiography in patients with GD (357). Myocardial damage can occur in thyrotoxic patients with congestive heart failure (358), even when coronary vessels are normal at baseline (359).

It has been suggested that the changes in the cardiovascular system are secondary to increased demand for metabolites and to increased heat production. Dilatation of superficial capillaries for the dissipation of heat does cause increased blood flow and cardiac output (360). However, the direct action of thyroid hormone on the heart is also increased, since the sinus node has higher intrinsic activity, the isolated thyrotoxic heart beats faster than normal, and isolated papillary muscle from a thyrotoxic heart has a shortened contraction time (361-363).

Historically, the cardiovascular effects of triiodothyronine have primarily been attributed to its modulatory influence on nuclear adrenergic receptor genes, among others (364, 365). However, more recent evidence has demonstrated a significant physiological role for the non-genomic effects of thyroid hormone, particularly in energy homeostasis (366-368). Cardiac muscle contractility is enhanced by a reduction in alpha-adrenergic and cholinergic receptors within the heart. This effect is coupled with an increase in beta-adrenergic receptors, heightened adenosine transport, and enhanced phosphorylation within myocardial cells. Additionally, there is an augmentation in cardiac Na+-K+ activated membrane ATPase activity, as well as an elevation in sarcoplasmic reticulum Ca++-activated ATPase activity. Finally, the increased synthesis of alpha-myosin heavy chains with increased ATPase activity also contributes to this heightened contractility (363, 369-371).

Fatourechi and Edwards used myocardial biopsy to investigate the presence of an autoimmune process in eleven cases of GD with low output cardiac dysfunction. Two patients had lymphocytic infiltrates suggestive of an autoimmune process, whereas the others did not, indicating that myocardial autoimmunity may occur but would not be the usual cause of cardiac dysfunction in thyrotoxicosis (372).

Table 4. Cardiovascular Manifestations in Graves' Disease
*Common:* · Palpitations · Tachycardia · Paroxysmal atrial tachycardia · Atrial fibrillation · Increased stroke volume and cardiac output. · Increased coronary blood flow and myocardial oxygen consumption · Decreased exercise capacity *Uncommon:* · Impaired cardiac reserve · Heart failure · Myocardial infarction

Table 4. Cardiovascular Manifestations in Graves' Disease

Common:

· Palpitations

· Tachycardia

· Paroxysmal atrial tachycardia

· Atrial fibrillation

· Increased stroke volume and cardiac output.

· Increased coronary blood flow and myocardial oxygen consumption

· Decreased exercise capacity

Uncommon:

· Impaired cardiac reserve

· Heart failure

· Myocardial infarction

Hematologic and Lymphatic Systems

In most patients the hemoglobin and hematocrit are in the normal or low-normal range (373). The glucose-6-phosphate dehydrogenase activity of red cells is increased in thyrotoxicosis (374), while blood volume and the red cell mass are also increased in some patients. In the past, severe thyrotoxicosis used to be associated with normocytic anemia with hemoglobin concentrations as low as 8 g/dl, likely related to iron deficiency and malnutrition (375, 376). However, the presence of anemia in a thyrotoxic patient these days requires a search for an additional explanation, other than thyrotoxicosis.

The reticuloendothelial and lymphocytic systems undergo hyperplasia. There may be generalized lymphadenopathy, and the thymus may be enlarged (377). The thymus enlargement should be considered when mediastinal abnormalities are noted on chest imaging; thymus enlargement resolves with resolution of thyrotoxicosis and that should inform the decision about possible biopsy (378). Relative lymphocytosis and neutropenia with a normal or slightly low total white cell count, constitute the characteristic blood findings of GD (379). A relative and an absolute increase in the number of monocytes has also been reported (380). Significant pancytopenia with leukocyte counts under 3x10⁹/l and neutrophiles under 2x10⁹/l rarely occur, and if unrelated to drug therapy, tend to recover with restoration of euthyroidism (381).

GD is often associated with mild thrombocytopenia, and occasionally with idiopathic thrombocytopenic purpura (382). This co-occurrence is thought to reflect the autoimmune pathogenesis of both diseases. Fourteen percent of patients with immune thrombocytopenic purpura are reported to have coincident GD. Mild thrombocytopenia may disappear spontaneously or with treatment of hyperthyroidism, or if severe, may respond to glucocorticoid therapy (383). Bone marrow examination may show normal or increased megakaryocytes. In addition, platelet life span can be shortened due to a more rapid clearing by the activated reticulo-endothelial system.

Usually, thyrotoxicosis results in a mild hypercoagulable state. Although rare, cerebral venous thrombosis has been reported in association with thyrotoxicosis, suggesting that occasionally the propensity for coagulation can lead to profound consequences (384). Mild prolongation of the prothrombin time and elevation of several coagulation factors (Factors VIII, XIII, IX, XI, Von Willebrand, fibrinogen, and plasminogen activator inhibitor 1, among others) are often seen with thyrotoxicosis, and return to normal with treatment. In addition, recent studies emphasize the role of thyroid hormones in promoting coagulation through non-genomic mechanisms involving platelet activation with subsequent endothelial interactions (385).

Gastrointestinal System

The appetite and the gastrointestinal track absorption are characteristically increased to try to offset nutritional requirements from the increase in catabolism seen in patients with thyrotoxicosis. Despite this, weight loss is usually predominant. In severe thyrotoxicosis, nausea, emesis, and abdominal pain can be present while intestinal transit time is decreased, and occasionally diarrhea occurs (386). Steatorrhea can be seen if fat intake is excessive. Achlorhydria can be as prevalent as 40% (387, 388). Gastric enzymes production is decreased, and a mild gastritis can be present (389). Fasting serum gastrin levels, and their responses to arginine, are increased (390).

Around 55% of patients with thyrotoxicosis can experience at least one abnormality in their liver blood tests. Alkaline phosphatase elevation is the most common, but other common abnormalities include hypoalbuminemia, mild prothrombin time elevation, elevated aminotransferases, hyperbilirubinemia, and elevated lactate dehydrogenase (LDH) (391). Mild to severe liver disease may be found, and the liver is frequently palpable regardless of the coexistence of heart failure (392). Jaundice is possible but often seen when there is significant cholestasis from severe thyrotoxicosis (393, 394). The cause of hepatic disease has been thought to be multifactorial in the setting of congestive hepatopathy from heart failure, malnutrition, previous or concomitant liver disease (i.e., infectious or autoimmune disease), and drug related liver injury, and possible liver ischemia from a mismatch between oxygen consumption and oxygen delivery to the liver (395-399). All gastrointestinal manifestations tend to improve with restauration of the euthyroid state.

Renal System

Polyuria and occasionally glucosuria are seen in uncomplicated thyrotoxicosis. Polyuria does not indicate insensitivity to vasopressin, for the kidney responds normally to vasopressin with an increase in concentration of urine (400). Glucosuria may reflect accelerated absorption of sugar from the intestine and glucose intolerance. The glomerular filtration rate and renal blood flow are on average increased, probably secondary to increased cardiac output and a direct effect of thyroid hormone on renal function (401). Hyperuricemia with hypercalcemia can be seen in severe thyrotoxicosis, but it rarely injures the kidneys. In addition, occasionally hyposthenuria and uremia occur (402, 403).

Female Reproductive System

Menstruation is characteristically decreased in volume, and rarely amenorrhea with a proliferative endometrium can happen. The menstrual cycle may be either shortened or prolonged. The relative importance of a primary action of excess thyroid hormone on the gonadotroph function is unclear. However, an alteration in pituitary LH production and subsequent ovulation is suggested (404). Hyperprolactinemia is more common in patients with hyperthyroidism than in healthy controls (405). Premature ovarian failure can occur in association with GD in patients with polyglandular autoimmune syndrome type 3 (222).

Fertility is decreased, but pregnancy can develop. The incidence of miscarriage, premature delivery, pre-eclampsia, and gestational heart failure are increased by maternal thyrotoxicosis (406-408). Reduced fertility and increased miscarriage rates are associated with autoimmune thyroid disease (AITD) and positive antibodies. High maternal thyroid hormone levels and/or high titer of TSab (by crossing the placenta in the 3^rd trimester) can lead to fetal thyrotoxicosis with suppressed fetal TSH, lower fetal weight, and fetal death (409). However, pregnancy often ameliorates the biochemistry and the symptoms of thyrotoxicosis due to GD. Unfortunately, relapse is prone to occur in the 3-4 months following delivery.

Male Reproductive System

Peripheral conversion of testosterone and androstenedione to estrone and estradiol is increased in both sexes during hyperthyroidism (293, 410). Thus, men can have elevated circulating levels of free estradiol (411, 412), and some might experience gynecomastia with ductal elongation and epithelial hyperplasia (413, 414). In addition, the slightly elevated LH in men with gynecomastia suggests hypothalamic insensitivity to feedback control and some peripheral unresponsiveness to LH (411).

Both Leydig cell and spermatogenic abnormalities may be present. Previous small series have described a 71% rate of loss of libido, 56% rate of erectile dysfunction, and 80% rate of low sperm counts (<40 millions) in thyrotoxic men (415, 416). In these patients, the total testosterone level was elevated, but because the sex hormone binding globulin level was also high, the free testosterone level was reduced and the response to hCG was blunted. The abnormalities normalize when the patients become euthyroid (417). In addition, RAI therapy can cause transient reductions in both sperm count and motility but do not seem to cause permanent effects with ordinary treatment doses (under 14 mCi, equivalent to around 500 MBq) (417).

Adrenal Function

There are no obvious clinical signs or symptoms of altered adrenal cortical function in thyrotoxicosis, but distinct biochemical changes have been detected. In thyrotoxicosis the adrenal cortex is often hyperplastic and exogenous glucocorticoids are cleared from the plasma and metabolized at an accelerated rate (418). Since plasma glucocorticoid levels are normal and their rate of metabolism is increased, total daily metabolism and excretion of 17-ketosteroids and 17 hydroxy-corticoids are usually increased (419, 420). Furthermore, there is a relatively increased excretion of 11-oxycorticoid metabolites (421), which are biologically inactive compounds. There is increased secretion of adrenocorticotropic hormone (ACTH) by the pituitary, and subsequently increased production of steroids by the adrenal gland in order to maintain a normal concentration of active steroids in the peripheral blood and in the tissues (422, 423). However, a reduced response to exogenous ACTH suggests that adrenal reserve is reduced (424), and it has been hypothesized that in severe thyrotoxicosis and in thyroid storm there may be an element of adrenal insufficiency.

An increase in the 5-alpha metabolite of testosterone (androsterone) and a relative decrease in the 5-beta metabolite (etiocholanolone) are seen in the urine of thyrotoxic patients (425, 426). Because administration of substantial amounts of androsterone depresses the level of serum lipids, Hellman et al. have hypothesized that this change in steroid metabolism may be a way in which thyroid hormone affects lipid metabolism.

Skeletal System

Patients with mild thyrotoxicosis can experience some degree of bone mass loss and increased fracture risk, irrespective of age or sex (427-429). Those with thyrotoxicosis extending over several years may develop severe osteoporosis (430, 431). Skeletal mass is augmented after restauration of euthyroidism (428, 429).

The thyroid hormone receptors α1 (TRα1), TRβ1, and TRβ2 are expressed in human osteoblasts and bone marrow stromal cells. Histomorphometry evaluations with tetracycline labelling demonstrates accelerated turnover of bone, calcium, and collagen, both in spontaneous hyperthyroidism and in female treated with excess thyroid hormone (298, 432, 433). Serum osteocalcin, carboxy-terminal-1-telopeptide, and alkaline phosphatase may increase in parallel with hormone levels (434-437). These tend to normalize with restauration of euthyroidism.

The serum calcium level is usually normal but may be sufficiently elevated to produce nausea and emesis (402), and rarely, renal injury (403, 438). Fecal and urinary calcium excretion is greatly augmented, but kidney stones are infrequent since there is a concomitant polyuria with increase in excretion of colloids that stabilize the calcium (433). Intestinal absorption of calcium is usually reduced (439). The hypercalcemia appears to be a direct manifestation of thyroid hormone action on bone metabolism and can lead to secondary hypoparathyroidism with reduced 1,25-dihydroxyvitamin D (440, 441), and elevated serum phosphorus secondary to increased renal resorption (438, 442). It has been proposed that the hypercalcemia can usually be corrected partially or totally by the administration of glucocorticoids, but data is inconclusive (442, 443). Treatment of thyrotoxicosis is certainly able to normalize these abnormalities (444).

Metabolism

METABOLIC RATE

The basal oxygen consumption in thyrotoxicosis, as measured by the basal metabolic rate (BMR), is elevated compared to a euthyroid person. In extreme thyrotoxicosis, the BMR may be double the standard (445-447). In addition, the total metabolic rate, which is the BMR plus the increments from work, food, or stress, is elevated.

Observations regarding energy expenditure are mixed. An increased cost of muscular work with less efficient coupling of oxidation and energy use in patients with thyrotoxicosis was reported many years ago by Plummer and Boothby (448)and Briard et al. (449) among others. However, recent studies suggest that the increase in energy expenditure caused by work is not altered in thyrotoxicosis (450).

CARBOHYDRATE METABOLISM

Intestinal carbohydrate absorption is accelerated, as is its removal from the plasma. After a standard oral glucose load is given, the thyrotoxic patient characteristically has an early and rapid rise in blood glucose concentration in 30 - 60 minutes followed by a rapid fall, so that by two hours the concentration is normal (451-453). In non-diabetic thyrotoxic patients there is increased insulin demand and there could be some resistance to the action of insulin. Fasting blood glucose levels are associated with double the normal insulin concentration (454), and insulin resistance has been found in vitro utilizing adipocytes from patients with untreated hyperthyroidism (455).

Diabetes may develop (in patients with prediabetes) or worsen with the development of thyrotoxicosis and is ameliorated or may disappear when thyrotoxicosis is treated (456). In these patients, there is increased basal hepatic glucose production and reduced ability of insulin and glucose to suppress hepatic glucose production, another marker of insulin resistance (457, 458).

LIPID METABOLISM

Hypocholesterolemia is associated with thyrotoxicosis. It may be produced without a distinct decrease in total body or liver cholesterol. Part of the cholesterol-lowering action of thyroid hormone relates to malnutrition and weight loss, and part may be simply a manifestation of hypermetabolism. There is an increase both in synthesis and degradation of cholesterol, but the balance results in lower steady-state concentrations in the serum (459-469). Thyroid hormone directly enhances conversion of cholesterol to bile acids and their excretion in the bile, disposing of 70-90% of the cholesterol formed in the body (462). They may also affect cholesterol metabolism by directly increasing the number of membrane surface low-density lipoprotein (LDL) receptors (463). Furthermore, hepatic lipogenesis is also strikingly increased, both by direct action of thyroid hormones and in response to increased insulin levels. Overall, the levels of LDL, HDL, and apolipoproteins are lowered (461, 465-470). Triglyceride levels tend to be normal or slightly elevated (464), and the clearance rate of infused triglycerides might be elevated (461, 465-470). Plasma leptin levels are normal (468), and non-esterified fatty acids are elevated (471).

PROTEIN METABOLISM

In thyrotoxicosis, protein formation and breakdown are both accelerated. Despite the increased protein turnover, there is an overall protein deficit (472, 473). Nitrogen excretion is increased, and nitrogen balance may be normal or negative, depending on whether intake meets the demands of increased catabolism. Catabolism of collagen is increased, and urinary hydroxyproline excretion is characteristically increased (474). While the exact mechanism behind the effect of thyroid hormone on protein metabolism is not fully elucidated, it is believed to be mediated by a combination of mitochondria-dependent cytoplasmic mechanisms, and modulation of nuclear genetic transcription of genes involved in protein metabolism (475-477).

VITAMIN METABOLISM

The absorption of vitamin A is increased and conversion of carotene to vitamin A is accelerated in thyrotoxicosis (478). The requirements of the body are likewise increased, and low blood concentrations of vitamin A may be found. In addition, requirements for thiamine and vitamin B6 are increased (479).

ACKNOWLEDGEMENT

Sincere gratitude is extended to Dr. Leslie DeGroot, the author of the previous version, for laying the foundation of this chapter and his enduring influence on the subject.

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Familial Hypercholesterolemia: Genes and Beyond

ABSTRACT

Genetic disorders resulting in familial hypercholesterolemia (FH) include autosomal dominant hypercholesterolemia (ADH), polygenic hypercholesterolemia, as well as other rare conditions such as autosomal recessive hypercholesterolemia (ARH). All of these disorders cause elevations in low-density lipoprotein (LDL)-cholesterol (LDL-C) and, as a result, greatly increase the risk of cardiovascular disease (CVD). Genetic loci involved in ADH include the LDLR, which codes for the LDL receptor (LDLR), APOB, which codes for apolipoprotein B-100 (apoB-100), the major protein component of LDL, PCSK9, which codes for Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), the low abundance circulatory protein that terminates the lifecycle of the LDLR, and apolipoprotein E (APOE), which is an important constituent of triglyceride rich lipoproteins. Importantly, a large percentage of people with the severe hypercholesterolemic phenotype do not possess a readily identifiable gene defect and many likely have polygenic hypercholesterolemia. Thus, identification of a specific genetic pathologic variant is not a necessary condition for the diagnosis of a genetic hypercholesterolemia. Several formal diagnostic criteria exist for FH and include lipid levels, family history, personal history, physical exam findings, and genetic testing. As all individuals with severe hypercholesterolemia are at high risk for CVD, treatment is centered on dietary and lifestyle modifications and early institution of lipid-lowering pharmacotherapy. Treatment should initially be statin-based, but most patients require adjunctive medications such as ezetimibe and PCSK9 blocking monoclonal antibodies. Three large cardiovascular outcome trials have shown a reduction in atherosclerotic CVD when ezetimibe or PCSK9 blocking monoclonal antibodies were added to a background of statin therapy and consequently have assisted in shaping international guidelines and consensus recommendations. Novel therapeutics recently developed, include: inclisiran – a small interfering ribonucleic acid (siRNA)-based gene-silencing technology that inhibits PCSK9 production, bempedoic acid – an inhibitor of adenosine triphosphate (ATP)-citrate lyase with a large cardiovascular outcome trial demonstrating a reduction in CVD in patients with statin intolerance and is now FDA approved for a wide range of patients including heterozygous FH and patients with prior CVD (secondary prevention) or those at high-risk for CVD (primary prevention) and elevated LDL-C, and evinacumab – a fully human monoclonal antibody inhibiting angiopoietin-like 3 (ANGPTL3) (FDA approved for homozygous FH only). Patients with extreme and unresponsive elevations in LDL-C will require more aggressive therapies such as lipoprotein apheresis and agents for the treatment of severe hypercholesterolemia such as microsomal triglyceride transfer protein (MTP) inhibitors and evinacumab.

INTRODUCTION

Genetic disorders resulting in familial hypercholesterolemia (FH) consist of autosomal dominant hypercholesterolemia (ADH), autosomal recessive hypercholesterolemia (ARH), and polygenic hypercholesterolemia. The genetic architecture of FH is more complex than previously recognized and in fact is now believed to be associated with at least nine different genes with thousands of variants, the details of which are beyond the scope of this chapter. But briefly, the term “autosomal dominant hypercholesterolemia” refers to those patients with dominantly inherited severe hypercholesterolemia – low-density lipoprotein (LDL)-cholesterol (LDL-C) greater than 190 mg/dL, who likely harbor mutations in genes regulating serum LDL levels. Historically, the more common causes of ADH include “classic” FH, which is a codominant disorder involving aberrations in the LDL receptor (LDLR), as well as other codominant forms of “nonclassical” FH, which involve defects in two other genes that regulate plasma clearance of LDL, apolipoprotein B (APOB), which is the main protein of LDL (a ligand for the LDLR), and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), which synthesizes a low abundance circulatory protein that limits the LDLR lifespan (1,2). Autosomal dominant forms of ADH include mutations in apolipoprotein E (APOE), which synthesizes the main protein of triglyceride rich lipoproteins and signal transducing adaptor family member 1 (STAP1), whose function in cholesterol homeostasis remains largely unknown (2,3). However, recent data have determined that mutations in STAP1 are not a causative factor in FH (4-7). An autosomal recessive form of FH, ARH, is very rare and results from pathogenic variants in LDLR accessory protein 1 (LDL-RAP1). Other forms may be due to defects in lysosomal acid lipase (LIPA), ATP- binding cassette sub-family G member 5 and 8 (ABCG5/8), and cholesterol 7 alpha-hydroxylase (CYP7A1) (2,8). Finally, a common form of FH is attributable to multiple variations in several genes each with minor effects on cholesterol regulation (more than 50 loci identified, as opposed to a single large effect as seen in the textbook version of FH). Polygenic causes are relatively common and likely explain many of the patients who are genotype negative. In fact, up to 50% of patients referred to lipid clinics for possible or probable HeFH have a polygenic basis (2). Additionally, only approximately 2% of patients with an LDL >190 mg/dL and no additional clinical or family history compatible with FH have a pathogenic variant in one of the FH genes (9,10). Thus, having an elevated LDL-C does not necessarily indicate that the patient has ADH due to a pathogenic variation in the LDLR, APOB, PCSK9, or APOE genes. All forms of FH result in very high levels of LDL-C and increase the risk of early and accelerated coronary artery disease (CAD) (8). Yet, FH remains vastly underdiagnosed and thus, undertreated, representing an extraordinary missed opportunity for maintenance of cardiovascular health and prevention of cardiovascular events. It has been estimated that less than 10% of the patients in the US with FH have been diagnosed (11,12). This chapter will largely focus on the canonical forms of FH involving the traditional genetic loci described above.

GENETICS

The LDLR is an 893-amino acid cell surface glycoprotein that binds and internalizes LDL particles, primarily in the liver. Mutations in LDLR (i.e., “classic” FH) give rise to nearly 90% of cases of clinical ADH (13). Over 2000 such mutations in LDLR have been identified, including deletions, insertions, missense, copy number variants, and nonsense mutations (2). FH patients can be homozygous (traditionally with a prevalence thought to be 1 in 1,000,000 but based on contemporary genetic studies, the prevalence is thought to be closer to 1 in 300,000), carrying mutations in both alleles encoding for LDLR, or heterozygous (traditionally with a prevalence thought to be 1 in 500, with newer data suggesting as frequent as 1 in 200), possessing mutations in only one allele (2,14). Homozygous FH (HoFH) should be suspected when LDL-C exceeds 400 mg/dL, whereas heterozygous FH (HeFH) should be suspected when LDL-C is greater than 190 mg/dL in adults and 160 mg/dL in children (8). Patients with HoFH can be true FH homozygotes, with two identical mutations in each allele, versus compound heterozygotes, with a different mutation in each allele. In addition, FH can result in elevated levels of lipoprotein(a) (Lp[a]) through an unclear mechanism, not necessarily linked to the dysfunctional LDLR pathway (15-19). Elevated Lp(a), which historically was thought composed of 30-45% cholesterol by mass and reported as part of the LDL-C laboratory measurement, amplifies the already increased risk of incident CAD seen in those with FH (19). A more contemporary study suggests this range of cholesterol content contributed by Lp(a) to be more heterogenous with a range of 9-57% with a median value of approximately 15-20% in individuals with elevated Lp(a) (20). A recent evaluation of Lp(a) levels in a Danish Lipid Clinic found that elevated Lp(a) levels were common, with approximately 27% of individuals fulfilling a clinical diagnosis of FH due in part to elevated Lp(a)(21). This data suggests the LPA gene should be considered in the realm of possible causes for phenotypic FH and emphasize the importantance of Lp(a) testing. It is also important to be aware of “founder effects” in some populations. Founder effects influencing the type and frequency of mutations causing FH are seen among Afrikaners, French Canadians, Ashkenazi Jews, Christian Lebanese, and some Tunisian groups. Slimane et al. estimated the prevalence of individuals with HoFH and HeFH in Tunisia to be 1:125,000 and 1:165, respectively (22).

“Nonclassical” FH, which phenotypically resembles classic FH in presentation and severity, involves dominantly inherited gene defects in APOB, PCSK9, and APOE, which code for proteins that modulate ligand-LDL/LDL-like receptor interaction (2,23,24). ApoB is the major protein constituent of LDL and acts as a ligand for LDLR. Mutations in ApoB (most commonly a single base change at a position near amino-acid 3,500) block the binding of LDL containing the apoB-100 to LDLR, resulting in severely elevated levels of LDL-C. This condition was originally defined as “familial defective APOB-100” or FDAB (25). PCSK9, on the other hand, is a circulating protein that terminates the lifecycle of LDLR by binding to it and targeting it to lysosomal degradation. Gain-of-function (GOF) mutations in PCSK9 lead to a FH phenotype, whereas loss-of-function (LOF) mutations lead to lower LDL-C and protection from coronary atherosclerotic events (26,27). Absence of circulating PCSK9 has been reported in a few subjects, who were reportedly healthy and had LDL levels around 20 mg/dL (28,29). Collectively, these observations spurred a frenzy of targeted research that led to the development and FDA approval of therapeutic antibodies against PCSK9 to reduce LDL levels in individuals with atherosclerotic cardiovascular disease (ASCVD) and/or in FH. Mutations in ApoE (an in-frame three base-pair deletion at position 167 in exon 4) block the binding of triglyceride rich lipoproteins (i.e., chylomicrons, chylomicron remnants, very low-density lipoprotein [VLDL-C], intermediate-density lipoprotein [IDL-C]) to the E receptor (belongs to the LDLR superfamily), and limits clearance of these particles from plasma (30). The prevalence of ADH resulting from mutations in APOB, PCSK9, and APOE has been difficult to estimate, but it is agreed that these are 5-10%, <1%, and <<1% respectively (2).

Finally, an additional ultra-rare recessive genetic disorder causing hypercholesterolemia bears mentioning. It involves a homozygous deletion mutation in the gene CYP7A1. This gene codes for the enzyme cholesterol 7α-hydroxylase, which catalyzes the initial step in cholesterol catabolism and bile acid synthesis. The mutation results in loss of enzymatic function and high levels of LDL-C, and was first identified in three homozygotes within a single kindred of English and Celtic descent (31). There are a number of other rare recessive genetic disorders that are associated with hypercholesterolemia, see table 1 below and other Endotext chapters on these rare genetic disorders (32,33).

TABLE 1. RARE GENETIC DISORDERS THAT CAN BE CONFUSED WITH ADH
Disorder	Description
Sitosterolemia	Autosomal recessive disorder due to mutations in ABCG5/8. Manifestations include hypercholesterolemia, marked elevations of plasma plant sterols, tendon and tuberous xanthomas, and premature ASCVD
Lysosomal acid lipase deficiency	Autosomal recessive disorder due to mutations in LIPA, which codes for lysosomal acid lipase. Manifestations include moderate hypercholesterolemia with depressed high-density lipoprotein cholesterol (HDL-C), accelerated atherosclerosis, and progressive liver disease.
Deficiency of LDL receptor adapter protein 1	Autosomal recessive disorder due to mutations in LDL-RAP1. Typically presents with very high LDL cholesterol levels.
Deficiency of cholesterol 7-alpha hydroxylase	Autosomal recessive disorder due to mutations in CYP7A1, which codes for the enzyme that catalyzes the first step in bile acid synthesis, resulting in high intrahepatic cholesterol and reduced surface expression of LDLR.

An important practical point is that 30-50% of people with the FH phenotype have no readily identifiable defects in any of the genes that have been mentioned here; thus, diagnosing an individual with the FH phenotype does not necessarily mean the presence of a monogenic defect in the LDLR pathway (34). The genetic confirmation for FH-causing mutations in ADH varies considerably (2,10). The rate of positive genetics is related to clinical criteria - in patients with definite FH based on clinical criteria 60-80% are positive whereas in patients with possible FH based on clinical criteria only 21-44% are positive (35). Additionally, the LDL-C level is crucial. When LDL-C is very high (i.e., >310 mg/dL), the frequency of monogenic pathogenic variants is as high as 92% (36). In patients without an identifiable pathologic genetic variant the etiology may be due to an as-yet-unidentified genetic error or to polygenic, epigenetic, or non-genetic factors, including co-morbid and environmental modifiers. For those without a mutation, an elevated LDL-C still confers elevated cardiovascular disease (CVD) risk (37). However, for any given LDL-C strata, those with a causal mutation compared to those without have higher risk for CVD, likely due to lifelong exposure to high LDL-C (10,38). In addition, unintended consequences of genetic testing (i.e., genetic discrimination for life or long-term care insurance and increased expense) must be taken into account. For these reasons, genetic testing should not be mandated, but should involve a shared-decision making model between patient and provider, encompassing benefits and risks of genetic testing, as well as patient values and preferences (39). On the other hand, others advocate for routine genetic testing citing as the rationale 1) facilitate a definitive diagnosis; 2) identify pathogenic variants with higher cardiovascular risk and therefore needing more aggressive treatment; 3) increase initiation of and adherence to therapy; 4) facilitate insurance approval for novel lipid-lowering therapies; and 5) cascade testing of first-degree relatives (25). Cascade screening on the basis of LDL-C alone (i.e., ≥190 mg/dL) has low sensitivity and specificity, however, identification of a pathogenic variant with genetic testing followed by cascade screening results in very high sensitivity and specificity. This is likely due to missed diagnoses of FH with reduced penetrance where LDL-C is <190 mg/dL (35). Nonetheless, the reduced costs and more widespread availability of genetic testing warrant performance of this test to obtain information that can help the physician familiarize with genotype-phenotype correlations and identify subjects that can be studied for the discovery of novel pathways leading to severe hypercholesterolemia. It must be noted that the FDA does not mention genetic testing as a measure to define FH (either heterozygous or homozygous).

PATHOPHYSIOLOGY

Most circulating LDL particles end up in the liver. The LDLR pathway is the predominant method for LDL uptake (40,41). ApoB binds to a specific binding site on LDLR and the receptor-ligand complex is subsequently internalized from clathrin-coated pits on the cell membrane. The receptor-ligand complex undergoes endocytosis and is targeted to the lysosome, where LDL is released for degradation while the LDLR is recycled back to the cell surface approximately 100-150 times in its 24 hour life cycle (2). PCSK9 terminates LDLR lifespan by disallowing its recycling, thus providing a physiologic mechanism of protein removal much different from, and stronger than, that caused by inducible degrader of LDL, an E3 ubiquitin ligase (26,42). There are other nonspecific and constitutively active pathways of LDL-C clearance as well (40,43). In HeFH, though transport through the LDLR pathway is reduced by 50%, LDL-C clearance is doubled through these other, non-LDLR pathways. The same holds true for HoFH, where despite a near-absolute reduction in LDLR transport, total LDL-C clearance via non-specific pathways is increased by 4-fold (44). Excess LDL-C, which accumulates in liver cells, is then re-exported via the apoB system back into the plasma, secreted into bile unchanged, or transformed into bile acids. This increased production of LDL adds to inefficient clearance via LDLR to cause elevated serum LDL levels typical of the FH phenotype (45).

ADH can thus be further classified into subtypes 1, 2, and 3, based on which protein of the LDLR pathway is causative (Figure 1). ADH-1 comprises mutations within LDLR, the canonical form of FH. There are six major classes of ADH-1 (see table 2 below), based on the type of mutation. These include those that: inhibit synthesis of LDLR; impede exit of mature LDLR from the endoplasmic reticulum; affect the binding site of LDLR to apoB-100; prevent the ligand-receptor interaction; prevent endocytosis of the LDLR-apoB-100 complex; or inhibit recycling of LDLR to the cell surface for further rounds of lipid uptake (not shown). ADH-2 comprises mutations of APOB that block the association of apoB-100 to LDLR. ADH-3 is due to GOF mutations of PCSK9, which reduce LDLR recycling and accelerate its lysosomal degradation (12). Some authors have suggested that mutations that affect binding of apoB-100 to LDLR carry a less severe phenotype than those that affect LDLR directly (46-50).

FIGURE 1 (24): ADH-1 comprises mutations within LDLR. There are six major classes of ADH-1, affecting: a) synthesis of LDLR; b) exit of mature LDLR from the endoplasmic reticulum; c) binding site of LDLR to apoB-100; d) endocytosis of LDLR-apoB-100 complex; and recycling of LDLR to the cell surface (not shown). ADH-2 comprises mutations in apoB that block the association of apoB-100 to LDLR. ADH-3 is due to GOF mutations of PCSK9, which reduce LDLR recycling and accelerate its lysosomal degradation. Adapted from Calandra et al. J. Lipid Research 2011; 52: 1885-926.

TABLE 2. THE SIX CLASSES OF LDLR MUTATIONS (51)
Class 1: synthesis of receptor or precursor protein is absent	The so-called null allele is a prevalent class of mutations and is generally associated with very high LDL-C levels. The molecular basis of this type of mutation shows a wide variety: point mutations introducing a stop codon, mutations in the promoter region completely blocking transcription, mutations giving rise to incorrect excision of mRNA, and finally, large deletions preventing the assembly of a normal receptor.
Class 2: absent or impaired formation of receptor protein	This class comprises mutations in which the normal routing through the cell is not complete or is only very slowly completed. Usually, there is a complete blockade of transport, and LDL receptors are unable to leave the ER. The Golgi apparatus is not reached, and the increase of 40,000 Da in molecular weight does not take place. Truncated proteins, as a result of a premature stop codon, and misfolded proteins, as a result of mutations in cysteine-rich regions leading to free or unpaired cysteine residues, are retained in the ER. However, quality control by the ER is not perfect, given the observation that sometimes misfolded proteins leave the ER but are processed more slowly. Such mutations give rise to class 2B mutations, in contrast to class 2A mutations that cause complete retaining in the ER.
Class 3: normal synthesis of receptor protein, abnormal LDL binding	Receptors characterized by this class of alleles show the normal rate of synthesis, exhibit normal conversion into receptor protein, and are transported to the cell surface, but binding to LDL is impaired. It is obvious that mutations in the binding domain underlie this class of receptors.
Class 4: clustering in coated pits, internalization of the receptor complex does not take place	The receptors in this class lack the property to cluster in coated pits (class 4A). This phenomenon, which makes interaction of receptors with the fuzzy coat impossible, is caused by mutations in the carboxyterminal part of the receptor protein. These mutated receptors are synthesized normally, folding and transport are normal, but clustering in coated pits is impossible, and sometimes the receptors are secreted even after they have reached the cell surface (class 4B).
Class 5: receptors are not recycled and are rapidly degraded	All mutations in this class are localized in the EGF-precursor homologous domain of the LDL receptor protein. This domain seems to be involved in the acid-dependent dissociation of the receptor-ligand complex in endosomes, after which the receptor can be recycled. When the entire EGF-precursor homologous domain is deleted by site-directed mutagenesis or when such a deletion occurs naturally in a homozygous FH patient, the receptor is trapped in the endosomes, and rapid degradation subsequently is observed.
Class 6: receptors fail to be targeted to the basolateral membrane	The class of mutations was recently discovered and is caused by alterations in the cytoplasmic tail of the protein. Such receptors do not reach the liver cell membrane and are probably rapidly degraded.

*Adapted from Gidding SS, et al. Circulation 2015;132:2167-92.

CLINICAL MANIFESTATIONS

Lipid Abnormalities in Heterozygous Familial Hypercholesterolemia

LDL-C levels are frequently greater than the 90^th percentile for age and gender. The magnitude of the LDL-C elevation is affected by the specific mutations causing FH with mutations in the LDLR leading to greater elevations in LDL-C levels than mutations in ApoB or PCSK9 (36,46-49,52). Null mutations in the LDLR are more severe than non-null mutations (53). Additionally, other genes that regulate LDL-C and environmental factors, such as diet, also influence the magnitude of the elevation in LDL-C (54,55). It should be recognized that a significant number of patients with genetically diagnosed FH have LDL-C <190 mg/dL. In some studies, approximately 50% of patients with genetically diagnosed FH have LDL-C levels <190 mg/dL (9,10). HDL-C and triglyceride levels are usually normal or only modestly altered (56-63). Elevated triglycerides and/or low HDL-C do not rule out the diagnosis of FH. Lp(a) levels are frequently elevated in patients with FH and may contribute to the increased risk of ASCVD (15-19). One should exclude secondary causes of marked elevations in LDL-C particularly hypothyroidism, renal disease, autoimmunity, and iatrogenic conditions (see chapter on Approach to the Patient with Dyslipidemia) (64). Another pertinent secondary cause that should be evaluated, particularly given escalating global rates of adiposity and the myriad treatment approaches at curtailing these trends, include dietary interventions that may induce a hyperlipidemic state. One such contemporary dietary fad that has garnered traction in the weight loss community is the ketogenic diet, which includes low level of carbohydrates (sufficient enough to induce the formation of ketones), moderate protein intake and high fat intake. It is well-documented that the ketogenic diet results in modest LDL-C elevations in most, but for some, can induce marked elevations in LDL-C, to the degree of mimicking a FH phenotype (65).

Atherosclerotic Cardiovascular Disease in Heterozygous Familial Hypercholesterolemia

Patients with FH have a 3-13-fold higher risk of ASCVD, and a 20-fold higher risk for early onset ischemic heart disease (66,67). Untreated males with FH have a 50% risk for a fatal or non-fatal myocardial infarction by 50 years of age whereas untreated females have a 30% chance by age 60 (68,69). However, it should be recognized that there is heterogeneity of ASCVD risk and much of this risk is potentially modulated by variance in genetic factors (70,71). Other cardiovascular risk factors, such as male sex, BMI, diabetes, hypertension, smoking, low HDL-C levels, and Lp(a) levels modulate the risk of ASCVD (66). Patients with FH who have corneal arcus or xanthomas are more likely to have ASCVD (72-74). Of particular note, patients with mutations that result in FH have a greater ASCVD risk than patients with equivalent LDL-C levels (10,38). This is likely due to the LDL-C elevations being present from birth (lifelong exposure to elevated LDL-C). This concept is termed "cholesterol-years", which tabulates the cumulative exposure, both degree of cholesterol elevation and duration of cholesterol elevation, as a measure of area under the cholesterol curve. Those with a greater exposure (higher cholesterol for longer) of cholesterol, will go on to develop atherosclerosis at a much more aggressive rate (75,76). This concept also highlights an important framework for early FH detection and treatment as vital interventions for optimal ASCVD risk reduction (76).

Other Manifestations

Early onset corneal arcus (age < 45) and tendinous xanthomas, particularly the Achilles tendon and dorsum of hands, are classical abnormalities that occur in patients with FH. Xanthelasma (xanthomas in eyelids) and tuberous xanthoma may also be seen. However, it should be recognized that in the modern era with the increased treatment of elevated LDL-C levels these abnormalities are no longer frequently seen (only 5-35% of patients have xanthoma or corneal arcus currently) (77,78).

Homozygous Familiar Hypercholesterolemia

This is a rare disorder with untreated LDL-C levels that vary but are markedly elevated (usually > 300 mg/dL but often > 500 mg/dL) (79). Patients who are LDLR negative have higher LDL-C and a poorer clinical prognosis than LDLR defective patients (79). Lp(a) levels tend to be higher than observed in patients with HeFH (16). Additionally, HDL-C levels tend to be decreased in HoFH (79). Tendinous xanthoma, tuberous xanthomas, and arcus cornea may appear in childhood (79). If untreated >50% of patient with HoFH develop clinically significant ACVD by age of 30 and cardiovascular events can occur before age 10 in some patients (80). Almost 90% of patients with HoFH suffered a cardiovascular event by age 40 (80). Cholesterol and calcium deposits can lead to aortic stenosis and occasionally to mitral regurgitation (79,81-83).

DIAGNOSIS

FH, despite its different underlying gene abnormalities, leads to severe hypercholesterolemia and a distinct FH phenotype with markedly increased risk of developing CAD. In general, there are five major clinical criteria for diagnosing FH (see table 3): a family history of early CAD (less than age 55 in a first-degree relative in men, and less than age 65 in women), early CAD in the index case, elevated LDL-C (greater than 190 mg/dL), tendon xanthomas (especially in the Achilles and finger extensor tendons), and corneal arcus (which is highly specific in younger patients, but overall an insensitive finding). Mutations in any of the aforementioned genes of the LDLR pathway, when they are identified, are diagnostic.

TABLE 3. MAJOR CLINICAL CRITERIA FOR DIAGNOSING FH
When to Suspect FH
1) If LDL-C levels are > 190 mg/dL (4.92 mMol/L) or non-HDL-C levels are >220 mg/dL (5.70mMol/L)
2) Patients with premature ASCVD (<55 years of age in male and <65 years of age in females)
3) Family history of hypercholesterolemia
4) When there is a positive family history of premature ASCVD (<55 years of age in male and <65 years of age in females)
5) When tendon xanthomas or corneal arcus (< age 45) are present on physical exam

As has been mentioned in other chapters of this text, when evaluating a patient suspected of having FH, it is critical to rule out secondary causes of hypercholesterolemia, such as hypothyroidism, nephrotic syndrome, and liver disease. Another extremely rare cause of non-FH has been described, involving autoantibodies to LDLR that inhibit receptor-mediated binding and catabolism of LDL-C (84).

There are three sets of statistically validated criteria that are most commonly used in the diagnosis of FH: the Dutch Lipid Network criteria, Simon Broome Register criteria, and Make Early Diagnosis to Prevent Early Deaths (MEDPED) criteria (85,86). These are summarized in Table 4, below (87).

TABLE 4. SCORING SYSTEMS FOR DIAGNOSING FH
MEDPED Criteria (USA)
	FH diagnostic if total cholesterol (LDL-C) levels exceed these cut points in mg/dL
Age	1^st degree relative		2^nd degree relative	3^rd degree relative	General population
< 18	220 (155)		230 (165)	240 (170)	270 (200)
20	240 (170)		250 (180)	260 (185)	290 (220)
30	270 (190)		280 (200)	290 (210)	340 (240)
> 40	290 (205)		300 (215)	310 (225)	360 (260)
Simon Broome Criteria (UK)
Total cholesterol (LDL-C) 290 (190) mg/dL in adults, or 260 (155) mg/dL in children	AND	DNA mutation Definite FH
		Tendon xanthomas in patient or 1^st or 2^nd Probable FH degree relative
		Family history of MI at age < 50 in 2^nd Possible FH degree relative or < 60 in 1^st degree relative OR Family history of total cholesterol > 290 mg/dL in 1^st or 2^nd degree relative
Dutch Criteria (Netherlands)
1 point	1^st degree relative with premature CVD or LDL-C > 95^thpercentile, OR Personal history of premature peripheral or cerebrovascular disease, OR LDL-C 155-189 mg/dL					Definite FH (8 points or more) Probable FH (6-7 points) Possible FH (3-5 points)
2 points	1^st degree relative with tendon xanthoma or corneal arcus, OR 1^st degree relative child (< 18 years) with LDL-C > 95^thpercentile, OR Personal history of CAD
3 points	LDL-C 190-249 mg/dL
4 points	Presence of corneal arcus in patient < 45 years old
5 points	LDL-C 250-329 mg/dL
6 points	Presence of a tendon xanthoma
8 points	LDL-C > 330 mg/dL, OR Functional mutation of the LDLR gene

Adapted from Fahed et al., Nutrition & Metabolism 2011;8:23.

Unlike MEDPED criteria, which use only lipid levels, the Simon Broome and Dutch criteria also use family history, personal history, physical exam findings, and genetic testing to establish an FH diagnosis. Again, however, it should be emphasized that FH should be diagnosed phenotypically, as opposed to genetically—most FH patients are genotype-negative and do not possess a clear genetic substrate for their hyperlipidemic phenotype, but they clearly warrant aggressive intervention.

It is important to note that in the modern era of earlier recognition, wide-spread statin use and possibly improved dietary messages, it is often difficult to make a definitive or probable diagnosis of FH using clinical criteria (i.e., treatment reduces the development of xanthomas and corneal arcus and reduces or delays the occurrence of ASCVD events in patients and relatives). Similarly, it is often very difficult to know the before treatment LDL-C levels (35).

While genetic screening is not required for clinical management, lipid screening in family members should be undertaken in all individuals by age 20, starting as early as age 2 (42). Cascade screening—i.e., lipid screening of first-degree relatives of the proband—is infrequently employed but is recommended as the most economical method of identifying new cases of FH (43). It is the responsibility of the examining clinician to attempt identification of other cases when making the diagnosis of FH in any given patient.

A potential novel solution to the underdiagnosis and undertreatment issues that plague the FH community lies in leveraging health information technology. The FIND FH study demonstrated the use of a machine learning algorithm can successfully utilize medical profiles within the electronic health record to consistently identify individuals with probable FH (88). This new approach possesses the promise of identifying FH patients on a national scale and will hopefully lead to increased initiation of effective preventive therapies and at an earlier time-point as well.

TREATMENT

Goals of Therapy

In genetic disorders causing hypercholesterolemia, aggressive lipid-lowering through lifestyle modification, pharmacologic treatment, and invasive treatments such as apheresis has been shown to decrease angiographically-apparent CAD and reduce cardiovascular events (80,89,90). However, traditional risk assessment tools like the Framingham risk score or pooled cohort equation do not apply to FH patients. Recent guidelines suggest that drug therapy should be initiated when LDL-C is greater than 190 mg/dL in all patients, including children over the age of eight (91,92). Most recommend at least a 50% reduction in LDL-C with initiation of high-intensity statin therapy as a starting goal, with some advocating for targeting LDL-C less than 100 mg/dL without ASCVD, less than 70 mg/dL with ASCVD, and even less than 55 mg/dL with ASCVD and at very high-risk (92-95). Additionally, consideration of non-statin options (ezetimibe, bile-acid sequestrants, bempedoic acid, and PCSK9 inhibiting [PCSK9i] therapeutics – including both monoclonal antibodies preferentially but also small interfering RNA should these thresholds not be met (92-94). The European Atherosclerosis Society suggests LDL-C goals of less than 135 mg/dL in pediatric patients, less than 100 mg/dL in adults, and less than 70 mg/dL in adults with known CAD or diabetes mellitus(11).

TABLE 5. GUIDELINE RECOMMENDATIONS FOR TREATING FH
	NLA Expert panel on pediatric FH(91)	AHA/ACC 2018 cholesterol guideline(92)and ACC non-statin ECDP(94)	NLA Expert panel on adult FH(93)	EAS guideline on FH(11)
Age to initiate treatment	≥ 8 years (earlier in special cases i.e., HoFH)	≥ 20 years	Not specified – “After a confirmatory diagnosis of FH …adult FH patients should receive initial treatment”	≥ 8 years
Agent recommended	Statins are preferred Non-statin options (ezetimibe, BAS, fibrates, niacin) are discussed but not routinely recommended due to lack of FDA approval or adverse drug events	Statins are preferred Non-statin options (ezetimibe, BAS, bempedoic acid, PCSK9i therapeutics) are also recommended as add on therapy	Statins are preferred Non-statin options (ezetimibe, BAS, niacin) or LDL apheresis are also recommended as add on therapy or in statin intolerant patients	Statins are preferred Non-statin options (ezetimibe, BAS) or LDL apheresis are also recommended as add on therapy or in statin intolerant patients
Goal of therapy	≥ 50% reduction in LDL-C or LDL-C < 130 mg/dL	≥ 50% reduction in LDL-C or LDL-C < 100 mg/dL in those without ASCVD, < 70 mg/dL with ASCVD, and < 55 mg/dL with ASCVD and at very high-risk	≥ 50% reduction in LDL-C	LDL-C < 135 mg/dL in pediatrics, < 100 mg/dL in adults, < 70 mg/dL in adults with CAD or diabetes

AHA / ACC: American Heart Association / American College of Cardiology; EAS: European Atherosclerosis Society; ECDP: Expert Consensus Decision Pathway; NLA: National Lipid Association; BAS: bile-acid sequestrants.

In our view in FH patients without clinical ASCVD one should aim for an LDL-C level <100 mg/dL (2.59 mMol/L). In FH patients with clinical ASCVD the goal, at a minimum should be an LDL-C level <70 mg/dL (1.81 mMol/L) with many experts recommending LDL-C levels <55 mg/dL (1.4 2mMol/L), particularly when patients have additional risk factors (acute coronary syndrome, diabetes, polyvascular disease, etc.) (96,97). In patients without ASCVD but who are at high risk due to other risk factors such as diabetes, Lp(a) >50 mg/dL, smokers, a strong family history of premature ASCVD, etc. many experts would recommend an LDL-C goal of <70 mg/dL (1.81 mMol/L). While LDL-C goals continue to decline, attainment of these goals is increasing more difficult and seldom attained in most FH patients.

Treatment of Patients with Heterozygous Familial Hypercholesterolemia

As with almost all metabolic disorders we should encourage the patient to follow a lifestyle that will reduce disease manifestations. However, lifestyle changes are very rarely sufficient to lower LDL-C levels to the desired range in patients with FH and therefore cholesterol lowering drugs will be required (see figure 2 and tables 6 and 7) (for detailed information on cholesterol lowering drugs see the chapter on cholesterol lowering drugs (98). In patients with HeFH, statins are a mainstay of treatment, despite the dearth of randomized clinical trials of statin efficacy focused on this special population. Statins are FDA approved for use in pediatric FH patients beginning at age 8-10 years for HeFH and in the first year of life or at initial diagnosis for HoFH (51,79,96,99-103). Data from longitudinal observational studies suggest statin initiation in childhood is both safe and effective, reducing LDL-C burden and corresponding atherosclerosis rates over follow-up of up to 20 years (51,104). The major early statin trials (4S and WOSCOPS) likely had study populations that were enriched with FH patients, given that mean baseline LDL-C ranged from 189 to 216 mg/dL (105,106). Patients should be treated with atorvastatin 40-80 mg per day or rosuvastatin 20-40 mg per day (i.e., high-intensity statin therapy). As monotherapy, statins can reduce LDL-C by up to 60% in HeFH patients but typically display a blunted response (10-25%) in HoFH patients depending on LDLR functionality (79,95,107). Updated guidance on statin therapy during pregnancy was issued by the FDA during a drug safety communication in 2021, removing the strongest warning against using statins during pregnancy (108). Though statins should still be avoided in most women attempting contraception during pregnancy, particular during the first trimester, the available evidence suggest that statins are likely not teratogenic. The updated guidance allows for more flexibility for treating pregnant women as part of a shared decision-making process, particular those at the highest ASCVD risk, including those with FH (109).

The vast majority of patients, however, require additional pharmacotherapy. When intensive statin therapy does not result in an LDL-C level in the desired range additional therapy should be added. Given that ezetimibe is generic, relatively inexpensive, well tolerated, and has evidence for ASCVD risk reduction in a large cardiovascular outcome trial, this is frequently the next drug used (110). One can anticipate that ezetimibe 10 mg per day added to intensive statin therapy will result in an approximate 20% further reduction in LDL-C. If this is not sufficient one can then use a PCSK9i monoclonal antibody to achieve the desired cholesterol goal. Adding a PCSK9i monoclonal antibody will result in a further 50-60% decrease in LDL-C levels and in most patients will result in LDL-C levels in the desired range. In some instances, if the LDL-C level is far from goal (>25%) on intensive statin therapy one can skip treating with ezetimibe and proceed directly to adding a PCSK9i monoclonal antibody. Bempedoic acid (discussed in more detail below) is an acceptable third- or fourth-line add-on agent if additional LDL-C lowering is needed. It is worth noting that bempedoic acid also comes as a combination product with ezetimibe providing approximately 40% LDL-C reduction in one tablet. This may be pertinent for patients with FH for whom pill burden is a real concern. In certain instances, bile resin binders may be useful in the treatment of FH (for example pregnant and lactating patients).

FIGURE 2. Approach to the Pharmacologic Treatment of Patients with Heterozygous Familial Hypercholesterolemia.

PCSK9 inhibitors consist of two therapeutic modalities, 1) monoclonal antibodies (alirocumab and evolocumab) that work extracellularly to sequester the PCSK9 protein, and 2) inclisiran, a novel, small interfering ribonucleic acid (siRNA)-based gene-silencing technology that inhibits mRNA translation and intracellular production of PCSK9 by the liver (111).

The effect of PCSK9i monoclonal antibodies in patients with HeFH has been extensively studied and consistently demonstrate potent LDL-C lowering on the order of 50-60% (63,112-126). An analysis of the ODYSSEY trials (FH I, FH II, LONG TERM, and HIGH FH) evaluated alirocumab use (75 or 150 mg subcutaneously every 14 days) in 1,257 HeFH patients. The primary endpoint was LDL-C at 24 weeks; alirocumab resulted in a more than 55% reduction in LDL-C compared with placebo (122). In another trial, alirocumab 150 mg every 14 days in 62 apheresis patients reduced the primary endpoint, rate of apheresis treatments over 12 weeks, by 75%, with 63.4% of patients completely discontinuing apheresis treatments due to well controlled LDL-C values (121). The RUTHERFORD-2 trial evaluated more than 300 patients with HeFH randomized to evolocumab (140 mg subcutaneously every 2 weeks or 420 mg subcutaneously monthly) versus placebo. At both dosing regimens, evolocumab resulted in significantly reduced LDL-C at 12 weeks compared with placebo (>60%) (63). In all trials, PCSK9i monoclonal antibodies were well tolerated, with most demonstrating treatment-emergent adverse events (TEAEs) similar to placebo. Clinically, the most common adverse events include: injection site reactions, mild cold or flu-like symptoms, nasopharyngitis, and myalgias (127). Thus, PCSK9i monoclonal antibodies are very effective at lowering LDL cholesterol levels and safe in HeFH patients.

Additionally, there are two cardiovascular outcomes trials that evaluated the FDA approved monoclonal antibodies targeting PCSK9. The FOURIER trial evaluated almost 28,000 subjects with stable vascular disease (CAD, stroke, peripheral arterial disease) on optimized statin therapy and randomized them to either placebo or evolocumab. Evolocumab therapy was associated with a 60% reduction in LDL-C and a 15% reduction in the primary 5-point major adverse cardiovascular event (MACE) rate (128). The FOURIER had an open-label extension trial (FOURIER-OLE) which followed 6,635 patients from the parent trial and allocated all patients (those originally assigned placebo or evolocumab) to evolocumab and followed over a median and maximal exposure of 7.1 years and 8.4 years respectively. Those originally assigned evolocumab in the parents study continued to display significant reductions in ASCVD events and even cardiovascular death as compared to those assigned placebo - establishing the important notion of a legacy effect with PCSK9i, where earlier initiation of therapy provides cardiovascular prevention that cannot be achieved when delayed. This study represents the longest follow-up to date of PCSK9i monoclonal antibodies and confirms that attainment of low LDL (30 mg/dL) is both safe and effective for preventing cardiovascular events (129).

ODYSSEY OUTCOMES enrolled approximately 18,000 subjects with recent acute coronary syndrome (1-12 months prior to enrollment in the trial) who were on high-intensity statin therapy at baseline and randomized them to placebo or alirocumab. Alirocumab was also associated with a 15% reduction in the primary outcome, in this case a 4-point composite of MACE (130). Major guidelines, consensus documents, and expert recommendations suggest consideration of a PCSK9 inhibitor (in addition to background statin +/- ezetimibe therapy) in high-risk patients with established ASCVD and/or in patients with severe hypercholesterolemia when LDL-C values exceed 55/70 or 100 mg/dL respectively (92,94,96,97,131-134). Both monoclonal antibodies have an FDA labeled indication for ASCVD risk reduction in patients with established ASCVD. Despite potent LDL-C lowering, a good safety profile, and robust clinical data demonstrating cardiovascular benefit, PCSK9i monoclonal antibodies are significantly underutilized among both patients with established ASCVD and those with FH, with reports suggesting only 1% to 2% of eligible patients currently prescribe PCSK9 inhibitors (135,136).

Inclisiran has been thoroughly studied in the ORION clinical development program, a series of phase 1 to 3 trials designed to investigate the pharmacokinetics, pharmacodynamics, optimal dose, efficacy, and safety of inclisiran in specific populations (111). ORION-9 was a phase 3, randomized, double-blinded, placebo-controlled trial evaluating use of inclisiran 300 mg given subcutaneously on days 1, 90, 270, and 540, in 482 HeFH patients with baseline LDL-C of ≥100 mg/dL(137). Treatment with inclisiran produced a placebo-corrected LDL-C reduction of 47.9% (P<0.001) at day 510. Response based on genotype was as expected, with LDL-C reductions of 41.2% to 59.2% for all except PCSK9GOF variants which were associated with dramatic LDL-C reduction of 89.7%. Pre-specified exploratory cardiovascular event (CV death, cardiac arrest, non-fatal MI, or non-fatal stroke) rates were comparable in both treatment groups (4.1% inclisiran vs. 4.2% placebo). Inclisiran was tolerated well during the trial with adverse events similar between treatment groups, with most adverse reactions mild to moderate in nature. The most common adverse event was injection site reactions, which was 10-fold higher in the inclisiran group (17%) compared to placebo (1.7%), however, 90% of these were graded as mild severity. Future studies will evaluate inclisiran in an adolescent HeFH population in the ORION-16 trial (138). Inclisiran was FDA approved in December 2021 as an adjunct to diet and statin therapy for the treatment of adults with primary hyperlipidemia, including HeFH, to reduce LDL-C(139).Pooled analyses of ORION-9 (patients with HeFH), ORION-10 (patients with ASCVD), and ORION-11 (patients with ASCVD or ASCVD risk equivalents) demonstrated durable LDL-C reduction of approximately 50% with a good safety profile with only injection site reactions, predominantly mild, and bronchitis as potentially drug-induced adverse effects (140,141). Additionally, prespecified exploratory analysis of MACE which was a non-adjudicated outcome and a safety event, and which should be regarded as hypothesis generating only, hinted at a reduction in atherosclerotic events with inclisiran (141). The question of whether inclisiran reduces cardiovascular events will be definitively determined with two large cardiovascular outcome trials – ORION-4 and VICTORIAN 2P, with estimated completion dates in 2026 and 2027 respectively (111). Though PCSK9 inhibition by monoclonal antibodies and inclisiran are comparable in LDL-C reducing capacity, there are important differences to acknowledge 1) inclisiran has a long biological half-life allowing for twice yearly dosing and possible medication adherence advantages, 2) PCSK9i monoclonal antibodies are administered by patients in their home and inclisiran is to be administered by a healthcare professional in a healthcare setting, and 3) while PCSK9i monoclonal antibodies have robust data proving reduction in ASCVD events, clinical outcome trials with inclisiran are still in progress. Additional considerations with the in-clinic administration of inclisiran include a new medication use process which includes procurement, storage, administration, and billing, which is a new workflow to the cardiovascular community and may take time to acclimate to. There are also the financial implications from the patient perspective as inclisiran is billed under medical insurance, not pharmacy benefits, which has the potential for reduced out-of-pocket expense for Medicare recipients specifically (142,143).

Novel approaches to inhibit PCSK9 action, termed third-generation PCSK9 inhibitors, are currently in various stages of clinical development. These include once orally administered macrocyclic peptide agents (i.e., MK-0616), monthly subcutaneous injection fusion protein binders (i.e., Lerodalcibep), CRISPR-based gene editing (i.e., VERVE-101), and PCSK9 vaccines (144). New PCSK9 modalities offer potential advantages such as oral administration, storage at room temp, and less frequent administration. The magnitude of LDL-C reduction with these technologies is similar to currently existing PCSK9 therapeutics, approximately 40%-60% lowering, except for vaccination, which appears less robust with approximately 10%-30% LDL-C reduction. Time will tell if and how these newer therapies position themselves among the ever-changing lipid-lowering landscape.

Bempedoic acid, a novel inhibitor of adenosine triphosphate (ATP)-citrate lyase (ACL), an enzyme upstream from 3–hydroxy–3–methylglutaryl coenzyme A (HMG–CoA) in the cholesterol synthesis pathway, is the newest orally administered lipid-lowering therapy (145). Bempedoic acid is a prodrug, that is converted into its active form (bempedoyl-CoA) by very long-chain acyl-CoA synthetase 1 (ACSVL1), which is expressed in hepatocytes but is undetectable in muscle. Development of this agent was designed to circumvent the myotoxicity commonly associated with historical lipid-lowering therapies, primarily statins. Bempedoic acid was FDA approved on February 21, 2020 for use in patients with established ASCVD and/or HeFH who require additional LDL-C lowering as an adjunct to dietary intervention and maximally tolerated statin therapy (146). Bempedoic acid was evaluated in over 3,600 patients in the phase 3 CLEAR program trials, of which, only 3.7% were HeFH patients (145). A pooled analysis from two phase 3 trials, CLEAR Harmony and CLEAR Wisdom, demonstrated a similarly modest placebo-corrected LDL-C reduction at 12 weeks of bempedoic acid treatment in the HeFH cohort (22.3%) as compared to the overall population (18.3%) (147). Overall bempedoic acid was well tolerated in the phase 3 trials but occurrence of TEAEs was higher in the HeFH cohort compared to those without HeFH but was not increased with bempedoic acid versus placebo. Similar reports of efficacy and safety were seen in an updated analysis of phase 3 data in HeFH as well (148). In a real-world analysis of bempedoic acid, which was enriched in patients with HeFH (64%) and statin intolerance (74%), the drug was associated with clinically meaningful LDL-C lowering (mean reductions 20.3% to 36.7%), but high rates of TEAEs (50%) and drug discontinuations (35.9%)(149). Bempedoic acid completed a large cardiovascular outcome trial in 2023 with publication of the CLEAR OUTCOMES (150). The trial enrolled 13,970 patients with established cardiovascular disease (70%) or at high risk for cardiovascular disease (30%) who were deemed statin intolerant. Over a median follow-up of 40.6 months, the mean baseline LDL-C was reduced from 139 mg/dL to a timed-averaged, placebo-adjusted reduction of 22 mg/dL in the bempedoic acid group. Is important to note there was a larger drop in LDL-C levels in the placebo arm than usually observed in clinical trials, which may have diluted the difference in LDL-C reduction between the 2 groups. Nevertheless, the primary endpoint which consists of a 4-point MACE including death from cardiovascular causes, nonfatal MI, nonfatal stroke, or coronary vascularization was reduced from 11.7% in the placebo group vs.13.3% in the bempedoic acid group, HR 0.87 (95% CI 0.79 to 0.96), with statistically significant reductions seen the non-mortality outcome measures. Interestingly, the greatest relative risk reduction was seen in the primary prevention cohort (32%) as compared to the secondary prevention cohort (9%), however the clinical significance of this remains to be seen. Bempedoic acid was well-tolerated with small increases in biomarkers such as serum creatinine, blood urea nitrogen, hemoglobin, aminotransaminases, and uric acid, and rates of gout (3.2% vs 2.2%), and cholelithiasis (2.2% vs 1.2%) (151). In response to the CLEAR OUTCOMES data, the FDA updated the label for bempedoic acid which now includes “to reduce the risk of myocardial infarction and coronary revascularization in adults who are unable to take recommend statin therapy, including those not taking a statin, with established ASCVD or at high risk for ASCVD” (146).

Treatment of Patients with Homozygous Familial Hypercholesterolemia

The initial therapy in patients with HoFH is identical to the treatment of patients with HeFH. However, statins and ezetimibe may prove relatively ineffective in the treatment of HoFH because the mode of action of these drugs largely depends on the upregulation of functional LDLR in the liver. In HoFH, measurable activity of both copies of the LDLR is absent or greatly reduced (80). Drug-induced LDL-C lowering is diminished by more than 50% comparing HeFH to HoFH – high intensity statins lower LDL-C 50-60% vs 10-25% and ezetimibe lowers LDL-C by 15-25% vs <10%, respectively (see table 6) (95,152).

In patients with HoFH, response to PCSK9i monoclonal antibodies varies depending on the specific gene defect. In the TESLA-B trial, 49 patients with HoFH were treated with evolocumab or placebo every four weeks for 12 weeks. LDL-C in the evolocumab treatment arm was significantly reduced by almost 31% compared with placebo. In addition to overall reduction in LDL-C, the trial investigators examined the treatment effect by LDLR mutation status. They found that the response to evolocumab aligns with the genetic cause of HoFH, with a greater reduction in LDL-C observed in subjects with two LDL receptor-defective mutations (i.e., abnormal receptor functionality in both alleles) when compared with those patients with even just one LDL receptor-negative mutation (i.e., nonexistent receptor functionality in one allele). Evolocumab was well tolerated among the HoFH patients (153). Similar results were seen in other HoFH trials examining the PCSK9i monoclonal antibody (alirocumab) and over longer follow up durations, providing confirmation of durable LDL-C efficacy and safety with this therapeutic class (154-156).

Inclisiran, dosed as 300 mg subcutaneously on days 1 and 90 (or 104 if PCSK9 level was suppressed by >70%), was evaluated in 4 HoFH patients enrolled in the ORION-2 trial (157). Patients were ages 50, 46, 23, and 29 years, 50% female, and had baseline LDL-C 540 mg/dL, 547 mg/dL, 614 mg/dL, and 189 mg/dL, respectively. All had biallelic causative genetic variants in LDLR, and all were on high-intensity statins and ezetimibe. Inclisiran-induced LDL-C reductions ranged 11.7% to 33.1% at day 90, and 17.5% to 37.0% at day 180 in three of the four patients, similar to results in prior trials of PCSK9i monoclonal antibodies in HoFH patients with residual LDLR function. One patient (LDLR c.681C/G [defective]) who had a history of hypo-responsiveness to both PCSK9i monoclonal antibodies (<20% lowering), also exhibited no change in LDL-C with inclisiran treatment. No adverse events were recorded over a 10 month follow up. A second, larger study of inclisiran use within the HoFH population was evaluated in a 2-part, phase 3 ORION-5 trial (158). Part 1 is a double-blind randomized study where patients were randomized in a 2-1 fashion to inclisiran and placebo respectively at days 1 and 90. Part 2 consisted of an 18-month open label single-arm extension trial in which all patients received inclisiran at day 180 and every 6 months thereafter until end of study at day 720. The study included 56 HoFH adults on stable lipid-lowering therapies including high-intensity statin 100%, ezetimibe 66.1%, and apheresis 35.7%. The median age was 42.7 years, 60.7% were women, and 67.9% had established ASCVD. The baseline mean LDL–C was 315.3 mg/dL with a higher value in the placebo group (356.7 mg/dL) as compared to the inclisiran group (294 mg/dL). Notably, more patients in the inclisiran group displayed the null/null LDLR genotypes which are known to produce blunted response to PCSK9 inhibition (27% versus 15.8%) – an important difference that may have confounded the treatment results. The primary endpoint, placebo–corrected percent change in LDL-C from baseline to day 150 was not statistically significant (-1.68%; P=0.90). The absolute placebo–corrected change in LDL-C level from baseline to day 150 was 6.47 mg/dL (P=0.87). The lack of statistically significant change in LDL values occurred despite a 60.6% reduction in plasma PCSK9 levels with inclisiran use. There were wide fluctuations in LDL-C reduction based on genotype: +26.6% with homozygous LDLR, -26.6% with compound heterozygous LDLR, and -22.5% with other genetic types. A post-hoc sensitivity analysis excluding patients with LDLR null/null genotypes and undergoing lipoprotein apheresis revealed greater reduction in LDL-C from 12.9% to 30%, indicating that inclsiran requires sufficient residual LDLR function to be effective within this population. There were no statistically significant changes in other proteins including apoB, non-HDL C, lipoprotein (a) and total cholesterol. Similar to prior evaluations of inclisiran, the medication was well-tolerated. The lack of positive treatment results from the ORION-5 cannot exclude a small sample size compared to those enrolled in the PCSK9i monoclonal antibody trials, an imbalance in genotypes in the present study, or differing mechanisms for PCSK9 intubation (159). The upcoming study of inclisiran in adolescent (ORION-13) HoFH patients, Which is estimated to be completed by December 2024, will shed further light on the use inclisiran in HoFH. (160).

Standard triple drug therapy (statin, ezetimibe, PCSK9 inhibitor) often does not result in a sufficient lowering of LDL-C due to the combination of the very high baseline LDL-C and the relative resistance of patients with HoFH to drug therapy. Currently, there are no published reports of bempedoic acid use within the HoFH population. Novel agents approved specifically for the treatment of severe hypercholesterolemia include microsomal triglyceride transfer protein (MTP) inhibitors and apoB-100 antisense oligonucleotides (ASO). MTP is involved in the transfer of lipid droplets to apoB as well as assembly and secretion of apoB-containing lipoproteins in the liver and gut. MTP inhibition thus reduces production and secretion of chylomicrons and VLDL-C. In one study, 29 patients with HoFH were treated with the MTP inhibitor lomitapide for 26 weeks and were followed until week 78. Average LDL-C reductions were 50% (to 166 mg/dl) at week 26, 44% (to 197 mg/dL) at week 52, and 38% (to 208 mg/dL) at week 78 (161). Real-world observational data of lomitapide from the LOWER registry suggest slightly less robust LDL-C lowering (33%) and LDL-C goal attainment (65.4% attaining LDL-C <100 mg/dL, 41.1% attaining LDL-C <70 mg/dL), likely a result of inadequate dose titration (mean dose 10 mg/d, range 5-40 mg/d) due to a high burden of adverse effects with nearly quarter of patients discontinuing treatment due to side effects (162). Though lomitapide displays potent LDL-C lowering capacity, use is limited as a result of a significant side effect profile consisting of severe gastrointestinal complications and hepatotoxicity risk, as well as high medication cost.

Anti-sense oligonucleotide (ASO) molecules bind to specific mRNAs and target them for degradation, reducing protein synthesis in the process. Mipomersen, which was removed from the market in June 2018, is an ASO that binds to apoB-100 mRNA and thus prevents the formation of apoB-100. Mipomersen results in decreased synthesis of apoB-containing lipoproteins, mostly VLDL-C, eventually leading to a drastic reduction of LDL-C levels in plasma. In one trial, 51 patients with either genetically defined HoFH, untreated LDL-C levels of >500 mg/dL plus xanthomas, or evidence of HeFH in both parents were randomized to placebo versus mipomersen for a treatment duration of 26 weeks. In the placebo group, baseline LDL-C was 402 mg/dL and declined to 390 mg/dL; in the treatment group, baseline LDL-C dropped from 440 mg/dl to 324 mg/dL (163).

Evinacumab is the newest addition to the lipid-lowering treatment armamentarium, receiving FDA approval in February 2021 as an adjunct to other LDL-lowering therapies for treatment of adults and pediatric patients originally ≥12 years of age with HoFH, with the age being reduced to ≥5 years of age in March 2023 (152). Evinacumab is a fully human IgG4 isotype monoclonal antibody targeting a novel, non-LDLR pathway for LDL-C lowering by inhibiting angiopoietin-like 3 (ANGPTL3) (164). ANGPTL3 inhibition overrides the inhibitory effect on lipoprotein lipase (LPL) and endothelial lipase (EL) increasing the activity of these enzymes, producing a panlipid-lowering effect of apo-B containing lipoproteins (with the exception Lp[a]), reducing lipoproteins by approximately 50% from baseline (152). Specifically, evinacumab promotes LDL-C lowering through EL-dependent VLDL-C processing and clearance by LDLR independent pathways, thereby decreasing formation of LDL-C from VLDL-C.(165) The magnitude of LDL-C lowering was seen in both HoFH and HeFH populations and was similar regardless of genotype. Serial coronary computed tomography angiography (CCTA) evaluations of two HoFH patients enrolled in the ELIPSE HoFH trial demonstrated reductions in coronary total plaque volume (TPV) of 76% to 85% over 10 months of treatment with evinacumab (166). Evinacumab is dosed at 15 mg/kg of body weight given over a 60 minute intravenous infusion (152). The drug is well tolerated with adverse effects being infrequent, mild, and transient, consisting primarily of injection site reactions, flu/cold-like symptoms, pain, and fatigue. The long-term safety of evinacumab remains unknown. A recent real-world analysis of evinacumab use across six US academic medical centers, demonstrated good tolerability, potent LDL-C lowering (50.8%), and LDL-C goal attainment (30.4% achieving LDL-C goals of <70 mg/dL) among 24 patients followed for up to 63 weeks (167). Additionally, a real-world analysis demonstrated that evinacumab can be a complementary therapy to apheresis. A single center US academic medical institution described the use of two patients undergoing apheresis for whom evinacumab was added achieving LDL-C lowering of 42% to 58%, and LDL-C goal attainment of <55 mg/dL in one patient. The study concluded that evinacumab presents a major advancement in the treatment of HoFH and will allow for improved LDL-C goal attainment. Evinacumab will be complementary to ongoing lipoprotein apheresis in many, but for those with less severe residual LDL-C elevation, may allow for reduced apheresis frequency or potentially replace its use altogether (168). The most recent study of evinacumab was published January 2024 and included an open-label 3-part series evaluating evinacumab use in pediatric patients age 5 to 11 years of age with HoFH (169). Part A was a pharmacokinetic study of 6 patients which determined that the 15 mg/kg intravenously administered dose utilized within the adult patient population was appropriate for pediatric patients. Part B was a phase 3, single-arm, 24-week open-label study assessing the efficacy, safety, and pharmacokinetics of evinacumab in 14 patients. Part C is an ongoing, phase 3, 48-week open-label extension study with a 24-week follow-up designed to assess the long-term safety and efficacy of evinacumab and will include all 20 patients from parts A and B. The mean age for patients in the part B trial was 9.1 years, 57.1% of which were females with 71% of patients having biallelic different LDLR variance, with 5 patient's carrying null/null alleles. Additionally, 11 of the 14 of patients already had aortic stenosis at this young age. Baseline LDL-C was 263.7 mg/dL, 85.7% were on any statin, 50% were on high-intensity statin, 100% were taking non-statins (none on PCSK9 inhibitor), 14.3% were taking lomitapide, and 50% were on apheresis. The primary endpoint, mean LDL-C percent change from baseline was reduced by 48.3% with an absolute LDL-C lowering of 131.9 mg/dL at week 24. LDL-C reduction was seen as early as week 1 with near full response witnessed by week 2. Response was independent of age, sex, ethnicity, LDLR genotype, or baseline apheresis use. Evaluation of other lipoprotein parameters included reductions of 41.3% in apoB, 48.9% in non-HDL-C, 49.1% in total cholesterol, and 37.3% in Lp(a). Similar to that seen in the adult population, evinacumab was well-tolerated with no treatment-related serious or severe TEAEs noted and no drug discontinuations due to adverse effects. Immunogenicity potential was minimal with only 1 patient developing treatment emergent antidrug antibodies but no neutralized antibodies. Evinacumab is poised to play a significant role in HoFH management as it offers one of the most potent LDL-C lowering capabilities among existing treatments. Use in HoFH will likely be as an add-on therapy after high-intensity statin, ezetimibe, and PCSK9 inhibition. However, potential barriers to use may include high cost, intravenous administration, and requirement for administration in a healthcare or home infusion setting. Future use of evinacumab may extend beyond HoFH and could entail use in other therapeutic areas such as HeFH and severe hypertriglyceridemia.

In patients in which drug therapy is either not successful at lowering LDL-C or not well tolerated one can consider lipoprotein apheresis or potentially even liver transplantation (170). The FDA has approved lipoprotein apheresis for subjects with CVD and LDL-C >200 mg/dL or without CVD and LDL-C >300 mg/dL (171). This threshold has been moved to 160 mg/dL, and more recently to 100 mg/dL with FH and ASCVD, thus increasing the target population for cholesterol dialysis at a time when arrival of stronger medications is curtailing patient entry into this therapeutic program. The process, which involves removing apoB-containing lipoproteins from plasma, is usually performed every two weeks and results in a 60-70% reduction of LDL-C and Lp(a) in the immediate post-procedure period, with time-averaged reductions of 20-50%. Levels tend to revert to baseline within two weeks. For more detailed information on lipoprotein apheresis see the Endotext chapter on this topic (172).

TABLE 6. COMMON LIPID-LOWERING TREATMENTS FOR FH
Agent	Niacin	BAS	Fibrate	Statin	Ezetimibe
FDA approval date	1997*	1973	1981† 1993	1987	2002
Administration	PO	PO	PO	PO	PO
Dosing	Daily	Daily	Daily	Daily	Daily
LDL-C lowering (HeFH)	10%-25%	15%-30%	10%-20%	20%-60%	15%-25%
LDL-C lowering (HoFH)	<10%	<10%	<10%	10%-25%	<10%
Lp(a) lowering	20-30%	N/A	N/A	N/A	N/A
Relative cost	+/++	++	+	+	+
Safety concerns	Flushing, moderate GI intolerance (abd pain, nausea, vomiting, peptic ulcer), hyperglycemia, gout, hepatotoxicity	Moderate GI symptoms (abd pain, constipation, bloating, nausea), hypertriglyceridemia, fat-soluble vitamin deficiencies	Myalgia, mild GI symptoms (abd pain, diarrhea), cholelithiasis, increased LFT	LFT elevation, myalgia, DM risk	Mild GI symptoms (loose stool, diarrhea, cramping), myalgia, increased LFT
Other consider-ations		High pill burden, separate from other meds (binding)	Primarily used for triglyceride lowering, renal dose adjustments	Usually well tolerated

Adapted from Warden BA, et al. Expert Rev Cardiovasc Ther. 2021;1-13.

*Approval date is for niacin extended-release. Niacin has been used clinically for hypercholesterolemia since the 1950’s.

†Gemfibrozil FDA approved in 1981, fenofibrate in 1993.

TABLE 7. ADVANCED LIPID-LOWERING TREATMENTS FOR FH
Agent	Lipoprotein apheresis	Lomitapide	PCSK9i*	Bempedoic acid	Evinacumab
FDA approval date	1996	2012	2015	2020	2021
Administration	IV	PO	SQ	PO	IV
Dosing	2-4x monthly	Daily	1-2x monthly	Daily	Monthly
LDL-C lowering (HeFH)	50-85% (acute) 23-50% (time-average)	N/A	50%-60%	15%-25%	49%
LDL-C lowering (HoFH)	50-85% (acute) 23-50% (time-average)	20%-50%	zero-30%†	Unknown	49%
Lp(a) lowering	50-75% (acute) 20-40% (time-average)	zero-30%	20-30%	N/A	N/A
Relative cost	++++	++++	+++	++/+++	+++++
Safety concerns	IV access issues, hypotension, vasovagal episodes, fatigue, bleeding, hypocalcemia, anemia	Severe GI intolerance (diarrhea, nausea, vomiting, abd pain, cramping), fat malabsorption, hepatic steatosis, hepatotoxicity (REMS)	ISR, flu/cold-like symptoms	Mild GI symptoms (diarrhea, abd discomfort), gout, tendon injury, transient lab changes (SCr, BUN, LFT, Hgb, HCT, uric acid)	Nasopharyngitis, ISR, flu-like symptoms, fatigue, pain, headache, rare hypersensitivity reaction
Other consider-ations	Lengthy and frequent treatments, need for patient travel, DDI (heparin, ACEi)	Significant DDI, renal dose adjustments	Access and cost issues	Improves glycaemia, option for patients with SAMS	Reduces all non-Lp(a) apoB-containing lipoproteins

Adapted from Warden BA, et al. Expert Rev Cardiovasc Ther. 2021;1-13.

*PCSK9i: represent PCSK9 blocking monoclonal antibodies and inclisiran, a novel small interfering ribonucleic acid (siRNA)-based medication.

†Response aligns with genetic cause of HoFH with no reduction was seen in those with null-null LDLR mutations.

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