Monogenic Disorders Altering HDL Levels

ABSTRACT

Very low HDL-C levels (<20mg/dL) may be due to severe elevations in triglycerides, very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, and certain medications such as anabolic steroids. Additionally, variants in multiple genes that each have a small effect but cumulatively lead to a decrease in HDL-C can result in very low HDL-C levels. Finally, rare monogenic disorders such as familial hypoalphalipoproteinemia, Tangier disease, and lecithin acyltransferase (LCAT) deficiency can lead to very low HDL-C levels. In this chapter we discuss the lipid abnormalities and clinical features of these monogenic disorders causing very low HDL-C levels. An elevated concentration of apo A-I and apo A-II is called hyperalphalipoproteinemia (HALP). HALP is classified as moderate (HDL-C levels between 80 and 100 mg/dL) or severe (HDL-C levels > 100 mg/dL). HALP is a heterogeneous condition caused by a variety of genetic and secondary conditions (for example ethanol abuse, primary biliary cirrhosis, multiple lipomatosis, emphysema, exercise, and certain drugs such as estrogens). In many individuals HALP has a polygenic origin. Monogenic HALP includes CETP deficiency, hepatic lipase deficiency, endothelial lipase deficiency, and loss of function mutations in SRB1. In this chapter we discuss the lipid abnormalities and clinical features of these monogenic disorders causing HALP.

LOW HDL CONDITIONS

The inverse relationship between HDL-C and ASCVD risk is well established but it should be recognized that while this association is consistently observed recent genetic and cardiovascular outcome studies suggest that this association is not causal (1). However, as discussed below major reductions in HDL-C induced by specific monogenic disorders may increase the risk of ASCVD.

Isolated low HDL-C levels can occur; however, it is more commonly found in association with hypertriglyceridemia and/or elevated apo B levels, typically as part of the obesity/metabolic syndrome (2). Patients with very low HDL-C (<20 mg/dL) in the absence of severe hypertriglyceridemia, very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, anabolic steroids, or a paradoxical response to PPAR agonists are very rare (<1% of the population) (3,4). These individuals may have a very rare monogenic disorder associated with marked HDL deficiency, including familial hypoalphalipoproteinemia, Tangier disease, and lecithin acyltransferase (LCAT) deficiency. Table 1 summarizes the genetic, lipid, and clinical features of these monogenic low HDL conditions. Inheritance is autosomal co-dominant with heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes (3). In some individuals the decrease in HDL-C can be polygenic i.e., variants in multiple genes that each have a small effect but cumulatively lead to a decrease in HDL-C (5).

Table 1. Characteristics of Monogenic Low HDL Syndromes
	Effected genes	Lipids	Clinical features
Familial hypoalpha-lipoproteinemia	apo A-I/apo C-III/ apo A-IV apo A-I/apo C-III apo A-I	Apo AI undetectable, marked deficiency in HDL-C, low – normal triglycerides, normal LDL-C	Xanthomas Premature ASCVD Corneal manifestations
Tangier disease	ABCA1	HDL species exclusively preß-1, HDL-C <5 mg/dL LDL-C low (half normal)	Hepatosplenomegaly Enlarged tonsils Neuropathy ASCVD (6-7^th decade)
LCAT deficiency	LCAT	HDL-C <10 mg/dL apo A-I 20-30 mg/dL <36% cholesteryl esters Low LDL-C Presence of Lp-X particles	FLD develop corneal opacities (“fish eye”), normochromic anemia and proteinuric end stage renal disease FED only develop corneal opacities

Inheritance is autosomal co-dominant with heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes (3). FLD- Familial Lecithin: Cholesteryl Ester Acyltransferase Deficiency; FED- Fish Eye Disease

Familial Hypoalphalipoproteinemia

Familial hypoalphalipoproteinemia is a heterogeneous group of apolipoprotein A-I (apo A-I) deficiency states. This disorder is the rarest cause of monogenic severe HDL deficiency (6). These various conditions are characterized by the specific apolipoprotein genes that are affected on the apo A-I/C-III/A-IV gene cluster (3). The genes for these 3 apolipoproteins (apo A-I, apo C-III, and apo A-IV) are grouped together in a cluster on human chromosome 11. In patients with apo A-I/C-III/A-IV deficiency, apoA-1 is undetectable in the plasma and is associated with marked deficiency in HDL-C, low triglyceride levels (due to apo C-III deficiency), and normal LDL-C levels (3). Heterozygotes have plasma HDL-C, apo A-I, apo A-IV, and apo C-III levels that are about 50% of normal (3). This condition is associated with aggressive, premature ASCVD. Additionally, there is evidence of mild fat malabsorption due to deficiency of apo A-IV. Patients with apo A-I/C-III deficiency have undetectable apo A-I and a similar lipid profile as those with apo A-I/C-III/A-IV deficiency (3). This condition is also associated with premature ASCVD. It is distinguished from the former by presence of planar xanthomas and absence of fat malabsorption (since apo A-IV is present). Familial apo A-I deficiency is itself a heterogeneous group of disorders associated with numerous Apo A-I mutations (3). Common manifestations include undetectable plasma Apo A-I, marked HDL deficiency with normal LDL-C and triglyceride levels, xanthomas (planar, tendon, and/or tubero-eruptive depending on the specific gene mutation), and premature ASCVD. Some forms of the disease are also associated with corneal manifestations, including corneal arcus and corneal opacification. One of the interesting manifestations of familial apo A-I deficiency is that levels of apo A-IV and apo E containing HDL particles are only modestly reduced, with preserved electrophoretic mobility and particle size (7).

It is notable that familial hypoalphalipoproteinemia is associated with an increased risk of premature ASCVD presumably due to the marked deficiency in Apo A-I and HDL. Given the increased ASCVD risk associated with Apo A-I deficiency, treatment is directed towards aggressive reduction of LDL-C and non-HDL-C levels and reducing other cardiovascular risk factors.

Some mutations in Apo A-I are associated with low HDL-C levels and hereditary amyloidosis and are the second most frequent cause of familial amyloidosis (6,8). Note that HDL-C levels are not always decreased in patients with familial amyloidosis secondary to Apo A-I mutations. The N-terminal fragment of the mutated protein is found in the amyloid fragments.

Tangier Disease

Tangier disease is due to mutations in the gene that codes for ATP-Binding Cassette transporter A1 (ABCA1) and is inherited in an autosomal co-dominant manner (9,10). Fredrickson first reported this condition in two patients who hailed from Tangier Island in the Chesapeake Bay, for which the disorder is named. ABCA1 facilitates efflux of intracellular cholesterol from peripheral cells to lipid poor A1, the key first step of reverse cholesterol transport (11). As such, this disorder is characterized by severe deficiency of HDL-C (HDL-C <5 mg/dL) and the presence of only thepreß-1 HDL fraction of HDL (10). The poorly lipidated Apo A-I is rapidly catabolized by the kidney. These patients also demonstrate moderate hypertriglyceridemia and low LDL-C levels (10). The decrease in LDL-C is likely due to absence of the transfer of cholesterol from HDL to LDL. Studies have also suggested that an increase in LDL uptake by the liver also occurs (12). The increase in triglycerides may be due to the failure of HDL to provide co-factors that increase lipoprotein lipase activity. Additionally, ABCA1 deficiency in the liver increases triglyceride secretion and hepatic angiopoietin-like protein 3 secretion which could inhibit lipoprotein lipase activity leading to an increase in triglycerides (12,13).

Since ABCA1 deficiency impairs free cholesterol efflux from cells, there is accumulation of cholesterol esters in many tissues throughout the body (10). Classically, patients present with hepatosplenomegaly and enlarged yellow-orange hyperplastic tonsils, however, a wide spectrum of phenotypic manifestations is now appreciated with considerable variability in terms of clinical severity and organ involvement (9,10). Peripheral neuropathies are also a common complication and may be relapsing-remitting or chronic progressive (9,10). Tangier disease patients appear to have an increased risk of premature ASCVD, though not as pronounced as those with familial hypoalphalipoproteinemia (3,9,14). When the non-HDL-C levels are greater than 70mg/dL patients with Tangier disease are at higher risk of ASCVD whereas when the non-HDL-C levels are less than 70mg/dL ASCVD is low (9). Less common complications include corneal opacities and hematological manifestations such as thrombocytopenia and hemolytic anemia (9,10).

Individuals who are heterozygous for ABCA1 mutations have HDL-C levels that are variable but approximately 50% of normal with normal levels of preß-1 HDL but a deficiency of large α-1 and α-2 HDL particles (10). Cholesterol efflux capacity in heterozygotes has been reported as ~50% of normal. A mutation in one ABCA1 allele has been associated with increased risk of ASCVD in some studies and with no increase in ASCVD risk in other studies (15-20). Different mutations in ABCA1 result in varying HDL-C levels and phenotypes, which might explain the difference in ASCVD risk (21).

While Tangier patients manifest characteristically low HDL-C and Apo A-I, this lipid/lipoprotein phenotype is not adequate to make the diagnosis. ABCA1 gene sequence analysis is the preferred test to make the diagnosis of Tangier disease (10). Alternatively, non-denaturing two-dimensional electrophoresis followed by anti-apo A-I immunoblotting demonstrates only preβ1-HDL.

Currently, there is no specific treatment for Tangier disease (10). In fact, HDL-C raising therapies such as niacin and fibrates have proven ineffective in patients with this condition (22). Even HDL infusion was not beneficial (23). The major clinical issue in Tangier patients is disabling neuropathy; however, there is no effective intervention to manage this complication (10). Aggressive LDL-C lowering and treatment of other risk factors for atherosclerosis is recommended (10).

LCAT Deficiency

LCAT is an enzyme that is bound primarily to HDL, with some also found on LDL (24,25). It facilitates cholesterol esterification by transferring a fatty acid from phosphatidyl choline to cholesterol (24,25). The hydrophobic cholesteryl esters are then sequestered in the core of the lipoprotein particles. LCAT is critical in the maturation of HDL particles. LCAT deficiency is an autosomal co-dominant disorder that manifests as either familial LCAT deficiency (FLD) or fish-eye disease in homozygotes (FED) (24,25). In FLD, mutations in LCAT lead to the inability of LCAT to esterify cholesterol in both HDL and LDL, whereas in FED, mutations in LCAT lead to the inability of LCAT to esterify cholesterol in HDL but the ability of LCAT to esterify cholesterol in LDL is preserved (24,25). Patients with FLD have virtually no cholesterol esters in the circulation while patients with FED have subnormal levels of cholesterol esters carried in apo B containing lipoproteins (24,25). Heterozygotes having decreases in HDL-C levels approximately midway between normal and homozygotes.

Individuals with FLD develop corneal opacities (“fish eye”), normochromic hemolytic anemia (due to cholesterol enrichment of red blood cell membranes), mild thrombocytopenia, and proteinuric end stage renal disease, which is the major cause of morbidity and mortality (24,25). The corneal opacities begin early in life and some patients may need corneal transplants. The rate of development of renal disease is variable but in a large cohort renal failure occurred at a median age of 46 years (26). Patients with FED generally only manifest corneal opacities (24,25).

The lipid and lipoprotein profile in patients with FLD usually demonstrates low HDL-C levels (frequently <10 mg/dL) (24,27). In one cohort patients with FED tend to have higher HDL-C levels but in a large systematic review HDL-C levels were similar in patients with FLD and FED (24,27). LDL-C levels tend to be low in FLD and FED while triglyceride levels are increased (24,27). Lipoprotein X (Lp-X) particles are present in patients with FLD but not in patients with FED (24). Lp-X is a multilamellar vesicle with an aqueous core. It is primarily composed of free cholesterol and phospholipid with very little protein (albumin in the core and apolipoprotein C on the surface) and cholesteryl ester.

Given the association of Lp-X and kidney disease only with FLD (and not FED) and animal studies demonstrating the nephrotoxicity of Lp-X, it is likely that increased levels of Lp-X results in renal dysfunction in patients with FLD (25,28). Lp-X particles accumulate in the mesangial cells in the glomerulus and are thought to induce inflammation and breakdown of the basement membrane leading to proteinuria. It is notable that after renal transplantation in patients with FLD there is recurrence of renal damage in the transplanted kidney (26).

It is unclear as to whether LCAT deficiency is associated with an increased risk of ASCVD (25,29). Atherosclerosis imaging studies have yielded divergent data and the number of patients with FLD or FED studied is limited (25,29). In one study carriers of FLD mutations (i.e., heterozygotes) had a decrease in ASCVD while carriers of FED mutations had an increase in ASCVD (30). This may have been due to higher LDL-C levels in the carriers of FED mutations (30).

Current management of FLD focuses on managing the renal dysfunction. The associated kidney disease is traditionally managed with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and a low-fat diet (25). Whether lipid lowering drugs are beneficial is unknown. Possible future therapies include enzyme replacement therapy with recombinant human LCAT, liver-directed LCAT gene therapy, small peptide or molecule activators of LCAT, and HDL mimetics (31,32). Infusions of recombinant human LCAT has improved the anemia and most parameters of renal function in a patient with FLD (33). Administration of CER-001, an apolipoprotein A1 (apoA-1)–containing HDL mimetic, has been shown to have beneficial effects on kidney and eye disease in a patient with LCAT deficiency (34).

Approach to the Patient with Low HDL-C Levels

When encountering a patient with very low HDL-C levels it is important to first determine if this is a new abnormality or has been present for a long time. If prior HDL-C levels are normal, this excludes a primary monogenic etiology. If the decrease in HDL-C is new, one should consider the possibility of very poorly controlled diabetes, inflammation, infections, malignancy, liver disease, paraproteinemia, anabolic steroids, or a paradoxical response to PPAR agonists. Marked hypertriglyceridemia can also lead to very low HDL-C levels.

In a patient with long-standing very low HDL-C levels without an identifiable secondary cause, one should consider a monogenic etiology. To evaluate potential monogenic causes, a detailed family history, with attention to HDL-C levels, is important. Obtaining lipid levels from relatives is very helpful. A focused physical examination, with particular attention to the skin, eyes, tonsils, and spleen may point to a specific monogenic disorder. Plasma apo A-I levels should be obtained. Individuals with familial hypoalphalipoproteinemia deficiency have undetectable plasma apo A-I. Patients with Tangier disease demonstrate very low apo A-I levels (<5 mg/dL). LCAT deficiency is associated with apo A-I levels that are low but substantially higher than the other monogenic etiologies. Patients with LCAT deficiency also have a higher ratio of free: total cholesterol in plasma and measurement of plasma free (unesterified) cholesterol can be helpful. Two-dimensional gel electrophoresis of plasma followed by immunoblotting with antibodies specific for apo A-I separates lipid-poor preß-HDL from lipid-rich–HDL and can be helpful in differentiating these disorders. Genetic analysis is indicated when a monogenic disorder is suspected.

HIGH HDL-C CONDITIONS (HYPERALPHALIPOPROTEINEMIA)

An elevated concentration of apo A-I and apo A-II is called hyperalphalipoproteinemia (HALP). HALP is classified as moderate (HDL-C levels between 80 and 100 mg/dL) or severe (HDL-C levels > 100 mg/dL). While it is well recognized that high HDL-C levels are associated with a decrease in ASCVD it should be noted that very high HDL-C levels are paradoxically associated with an increase in ASCVD (35,36).

HALP is a heterogeneous condition caused by a variety of genetic and secondary conditions (for example ethanol abuse, primary biliary cirrhosis, multiple lipomatosis, emphysema, exercise, and certain drugs such as estrogens). In many individuals, the very high HDL-C levels have a polygenic origin (5,37). Given the focus of this chapter, monogenic causes of HALP will be reviewed. Monogenic HALP includes CETP deficiency, hepatic lipase deficiency, endothelial lipase deficiency, and loss of function mutations in SRB1. Despite epidemiology that demonstrates an inverse relationship between HDL-C and ASCVD risk, some forms of familial HALP are paradoxically associated with increased cardiovascular risk.

HALP is generally identified incidentally after routine assessment of a lipid profile as it is not usually associated with any signs or symptoms. Generally, patients are asymptomatic and no medical therapy is required.

Cholesterol Ester Transfer Protein (CETP) Deficiency

CETP transfers cholesteryl esters from HDL particles to triglyceride rich lipoproteins and LDL in exchange for triglycerides (11). Individuals who are homozygous for CETP variants have very high HDL-C levels (>100mg/dL) while heterozygotes have moderately increased HDL-C levels (38-41). LDL-C and apo B levels may be normal or modestly decreased. The increase in HDL cholesterol are largely due to the accumulation of cholesterol esters (39). The decrease in LDL-C is due to the failure of cholesterol ester transport from HDL to apo B containing lipoproteins. There is a predominance of small LDL particles. Individuals who are heterozygotes for CETP mutations show modestly elevated HDL-C levels (38,39). In Japanese individuals with HDL-C levels > 100mg/dL 67% were demonstrated to have CETP gene mutations (42). CETP deficiency is the most important and frequent cause of HALP in Japan. CETP deficiency is common in other Asian populations but is relatively rare in other ethnic groups (39). Despite extensive studies the effect of CETP variants on the risk of ASCVD is uncertain (38-40,43). A variety of studies have indicated that a decrease in LDL-C and non-HDL-C levels (i.e. pro-atherogenic lipoproteins) rather than an increase in HDL-C induced by CETP variants underlies a potential beneficial effect on ASCVD (44).

Endothelial Lipase (EL) Deficiency

Endothelial lipase (EL) is encoded by the LIPG gene and hydrolyzes phospholipids on HDL resulting in smaller HDL particles that are more rapidly metabolized (11). Genetic variants in LIPG have been identified Iin individuals with elevated HDL-C levels (38,39). As one would predict large HDL particles enriched in phospholipids are observed in individuals deficient in EL (39). Whether variants in LIPG leading to decreased EL activity and increased HDL-C levels reduces ASCVD risk is uncertain (38-40).

Hepatic Lipase (HL) Deficiency

Hepatic lipase (HL) is encoded by the LIPC gene and mediates the hydrolysis of triglycerides and phospholipids in intermediate density lipoproteins (IDL) and LDL leading to smaller particles (IDL is converted to LDL; LDL is converted from large LDL to small LDL) (11). It also mediates the hydrolysis of triglycerides and phospholipids in HDL resulting in smaller HDL particles (11). Several case reports of families with elevated HDL-C levels (HALP) caused by a genetically defined HL deficiency have been described (39,40). HL deficiency may also be associated with elevated triglycerides and cholesterol with increased intermediate density lipoproteins (IDL) (40,45). Several HL deficient individuals had premature ASCVD likely due to increased levels of apo B containing lipoproteins (40,45). Heterozygotes do not appear to have discrete lipoprotein abnormalities (45).

Scavenger Receptor Class B Type I (SR-BI)

Scavenger receptor class B type I (SR-BI) is encoded by the SCARB1 gene and facilitates the selective uptake of the cholesterol esters from HDL into the liver, adrenal, ovary, and testes (11). In macrophages and other cells, SR-B1 facilitates the efflux of cholesterol from the cell to HDL particles (11). SR-B1 deficient mice have an increase in atherosclerosis despite elevated HDL-C levels (46). Mutations in SCARB1 associated with decreased SR-B1 have been observed in individuals with high HDL-C levels (47-49). Heterozygotes have intermediate elevations of HDL-C between wild-type and homozygous individuals. Studies have suggested that some but not all mutations in SCARB1 result in an increased risk of ASCVD despite increased HDL-C levels (40,49). A decrease in adrenal function has been reported in some individuals with SCARB1 mutations likely due to a reduced ability of SR-B1 to facilitate cholesterol uptake into the adrenal glands (48,50). Abnormalities in platelet function have also been observed in some patients (50).

ACKNOWLEDGEMENTS

This work was supported by grants from the Northern California Institute for Research and Education.

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Pharmacological Causes of Hyperprolactinemia

ABSTRACT

Hyperprolactinemia represents a multifaceted endocrine disorder with both physiological and pathological causes. The increased use of anti-psychotic and anti-depressant medications has increased the role pharmaceutical agents play in inducing hyperprolactinemia, being the most frequent cause of hyperprolactinemia in clinical practice. This has particularly impacted females, who demonstrate a higher susceptibility to drug-induced hyperprolactinemia. Of these medications, anti-psychotics, neuroleptic-like medications, anti-depressants, and histamine receptor type 2 antagonists, emerge as the most prominent culprits. Furthermore, opioids, some anti-hypertensive agents, proton pump inhibitors, estrogens, and other less potent hyperprolactinemia-inducing medications are recognized as potential contributors to drug-induced hyperprolactinemia. Many herbal medicines are reported as lactogenic, but their ability to cause hyperprolactinemia remains unclear. This review endeavors to elucidate the intricate mechanisms underlying the induction of hyperprolactinemia by pharmacological agents. We have included available data on the prevalence and extent of drug-induced changes in prolactin levels. We have also included data on herbal agents. We have highlighted where controversial data are identified. Although a detailed exploration of how these medications impact prolactin regulation is beyond the scope of this chapter, this review aims to deepen our understanding of the interplay between pharmacological agents and their effects on prolactin levels, contributing to valuable insights, refined therapeutic approaches, and better patient care.

INTRODUCTION

The most common cause of consistently high prolactin levels is drug-induced hyperprolactinemia. The overall incidence is higher in women compared to men. Drug-induced causes tripled during the 20-year follow-up period, reflecting the increased prevalence of psychoactive drug use (1). Several drugs have been reported to induce hyperprolactinemia, either by inhibiting dopamine receptors or their actions or by directly stimulating prolactin secretion (2).

High levels of prolactin can be attributed to various physiological factors, such as pregnancy and breast-feeding, while minor increases in prolactin levels may also occur during ovulation, after sexual intercourse, during periods of stress, exercise, after food intake, or in association with irritation of the chest wall and breast stimulation. Pathological causes can be related to hypothalamo-pituitary disorders or non-hypothalamo-pituitary disorders. Hypothalamo-pituitary disorders include prolactin-secreting pituitary tumors (including lactotroph tumors, mammo-somatotroph tumors, mature pluri-hormonal PIT1-lineage tumors, immature PIT1-lineage tumors, acidophil stem cell tumors, multi-hormonal pituitary tumors, mixed somatotroph, and lactotroph tumors, and pluri-hormonal tumors) (3); hypothalamic and pituitary stalk compression or damage (non-prolactin-secreting pituitary adenomas, craniopharyngiomas, meningiomas, germinomas, granulomas, metastasis, Rathke cleft cysts, hypophysitis, radiation, surgery, and trauma); infiltrative pituitary disorders; pituitary hyperplasia (McCune-Albright, Carney complex, X-LAG). Other causes include primary hypothyroidism; adrenal insufficiency; systemic diseases such as chronic renal failure and liver cirrhosis; polycystic ovary syndrome; neurogenic causes (chest wall trauma or surgery, herpes zoster); seizures; untreated severe phenylketonuria; pseudocyesis (false pregnancy); autoimmune diseases (lupus, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, Behcet’s disease, polymyositis); cancers (breast, ovarian, colon, hepatocellular) (4). During the diagnostic process of hyperprolactinemia, it is crucial to consider the possibility of macroprolactinoma and the ‘hook’ effect, although the latter is usually not relevant in drug-induced cases (5).

Drug-induced hyperprolactinemia is often characterized by prolactin levels ranging from 25 to 100 ng/mL (530-2130 mIU/L). However, certain medications including metoclopramide, risperidone, amisulpride, and phenothiazines can lead to prolactin levels surpassing 200 ng/mL (4255 mIU/L) (6). On similar doses of prolactin-raising anti-psychotics, women with chronic use are more likely to develop hyperprolactinemia than men, reaching significantly higher prolactin levels, with mean levels of 50 ng/mL (1065 mIU/L) (7,8). Younger age was associated with higher prolactin levels in women, but not in men (8,9). Route of drug administration is important, with prolactin levels returning to normal after cessation of the drug: within 2-3 weeks after stopping oral treatment, but no sooner than 6 months after discontinuation of intramuscular depot administration (10).

This chapter will encompass a comprehensive discussion of all pharmacological causes as well as some alternative factors contributing to changes in prolactin levels especially hyperprolactinemia.

EPIDEMIOLOGY

Drug-induced hyperprolactinemia is the most common cause of consistently high prolactin levels. A retrospective follow-up study conducted in Scotland, involving 32,289 hyperprolactinemic individuals from 1993 to 2013, concluded that within the non-pregnancy-related group, the most prominent cause was drug-induced hyperprolactinemia (45.9%), followed by pituitary disorders (25.6%), macroprolactinoma (7.5%), and hypothyroidism (6.1%). Nevertheless, 15% of cases were deemed idiopathic. The overall incidence was higher in women aged 25-44 years old compared to men (1). Female predominance is reported in other studies with a female: male ratio of 5.9:1 and the mean age at diagnosis of hyperprolactinemia is 40 (range 14–85) years (2,11).The position of hyperprolactinemia as a side effect of medications has been assessed in a French Pharmacovigilance Database from 1985 to 2000, which reported 159 cases of hyperprolactinemia out of 182,836 adverse drug reactions (11). The rates of hyperprolactinemia related to therapeutic drug classes were recorded as 31% associated with anti-psychotics, 28% with neuroleptic-like drugs (medications with a similar mechanism of actions as neuroleptics, but used for different purposes, for example movement disorders or anti-emetics), 26% with anti-depressants, 5% with histamine receptor type 2 (H2-receptor) antagonists, and 10% with other drugs.

PROLACTIN CONTROL MECHANISMS

Prolactin is a polypeptide primarily produced in the anterior pituitary gland, with secondary production occurring in other tissues such as the gonads, mammary gland, endometrium, prostate, lymphocytes, hematopoietic cells, skin, brain, retina, inner ear cochlea, decidua, pancreas, liver, endothelium, and adipose tissue (12–14). In breast and prostate cancer, prolactin has even been proposed as a tumor marker (15,16). Prolactin acts through prolactin receptors (PRLR), which belong to the family of cytokine receptors associated with the non-receptor tyrosine Janus kinase 2. PRLR can activate the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway, MAPK (mitogen-activated protein kinase), PI3 (phosphoinositide 3-kinase), Src kinase, as well as the Nek3 / Vav2 / Rac1 serine / threonine kinase pathway (17). There are different isoforms of this receptor: a long isoform, intermediate isoform, 2 short isoforms S1a and S1b which are formed by alternative splicing and partial deletion of exons 10 and 11, and soluble PRLR (18). These different isoforms are expressed in different tissues, mostly studied in rats. The long isoform is mainly expressed in the adrenal glands, kidneys, mammary glands, small intestine, bile ducts, choroid plexus, and pancreas whereas the short isoform is in the liver and ovaries (19). Prolactin possesses nearly 300 functions apart from lactation including neuroprotection and neurogenesis, offspring recognition by both parents, adipose and weight homeostasis, islet functions, immune regulation, angiogenesis, osmoregulation, and mitogenesis (20).

The secretion of prolactin produced by lactotroph cells in the anterior pituitary gland has a circadian rhythm with higher levels during sleep and lower levels during wakefulness (21). Even though pulsatility frequency does not significantly change over 24 hours, the amplitude of pulses is higher during night and day sleep, while wakefulness is associated with an immediate offset of active secretion. Prolactin is lower during the rapid eye movement stage of sleep (22).

The synthesis and secretion of prolactin is under the complex control of peptides, steroid hormones, and neurotransmitters, which can act as inhibitory or stimulatory factors, either by a direct effect on lactotroph cells or by indirect pathways through inhibition of dopaminergic tracts, and are widely studied in mammals (2). Dopamine plays a crucial role in inhibiting prolactin secretion. Dopamine can bind the five types of dopamine receptors (G-protein coupled receptors): DRD1, DRD2, DRD3, DRD4 and DRD5, while lactotroph cells express mainly D2 receptors. Dopamine can reach the pituitary via three pathways (Figure 1): through the tuberoinfundibular dopaminergic (TIDA) system, the tuberohypophysial tract (THDA), and the periventricular hypophyseal (PHDA) dopaminergic neurons (23). TIDA neurons originate from the rostral arcuate nucleus of the hypothalamus and release dopamine into the perivascular spaces of the medial eminence and through long portal vessels dopamine reaches the anterior pituitary gland. The THDA neurons originate in the rostral arcuate nucleus and project into the medial and posterior pituitary lobes and release dopamine at these sites. From THDA tract dopamine then reaches lactotroph cells through the short portal vessels (24). PHDA neurons originate in the periventricular nucleus and axons terminate in the intermediate lobe and dopamine release follows the same direction as from the THDA neurons. Prolactin-inhibiting neurons are considered to be a functional unit working synchronously (23). The binding of dopamine to D2 receptors on the plasma membrane of lactotroph cells inhibits prolactin protein, PRL gene transcription, as well as lactotroph proliferation (24). The release of prolactin through exocytosis of prolactin secretory granules is influenced by dopamine through various pathways. Specifically, D2 receptors are coupled with pertussis toxin-sensitive G proteins, which subsequently inhibit adenylate cyclase activity, resulting in decreased levels of cyclic adenosine monophosphate (cAMP) (25).

Additionally, the activation of potassium (K+) channels occurs, leading to a reduction in voltage-gated calcium (Ca2+) currents and inhibition of inositol phosphate production. Collectively, these intracellular signaling events culminate in a decrease in the concentration of free calcium ions (Ca2+) resulting in membrane hyperpolarization, ultimately inhibiting the exocytosis of prolactin from its granules (26). The inhibition of PRL gene transcription occurs when D2 receptors are activated, leading to the inhibition of MAPK or protein kinase C pathways. This activation results in a reduction of phosphorylation events on Ets family transcription factors. These transcription factors play a crucial role in the stimulatory responses of thyrotropin-releasing hormone (TRH), insulin, and epidermal growth factor (EGF) on prolactin expression. Moreover, the Ets family transcription factors interact with the PIT1 protein, which is essential for cAMP-mediated PRL gene expression (27). Dopamine exerts anti-mitogenic effects by activating D2 receptors through multiple pathways. These include the inhibition of MAPK (mitogen-activated protein kinase) signaling, protein kinase A signaling, and stimulation of phospholipase D activity. Additionally, dopamine engages a pertussis toxin-insensitive pathway, activates the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, and inhibits the AKT/protein kinase B pathway (28–31).

In addition to the dopaminergic inhibitory system, the γ-aminobutyric acid (GABA)-ergic tuberoinfundibular system, culminating in the median eminence, exhibits inhibitory properties, albeit of lesser potency compared to the dopaminergic system, while also having a role in prolactin modulation. GABA-B receptors are discernible both within the anterior pituitary gland, contributing to the maintenance of low prolactin levels, and in TIDA neurons which can be powerfully inhibited by GABA via hyperpolarization, consequently contributing to an elevation in prolactin levels (32,33).

Prolactin itself has two negative feedback effects: through ``short-loop feedback regulation`` it enhances the activity of TIDA neurons, where both long and short forms of the PRLR are expressed, with the long isoform being predominant in the arcuate and periventricular nuclei, regulating tyrosine hydroxylase (a rate-limiting enzyme in dopamine synthesis) leading to increase of dopamine release, which inhibits prolactin, as well as autocrine inhibition (34,35). Several other local factors influence prolactin release within pituitary gland as shown in Figure 1.

Figure 1. Prolactin – Central Nervous System Regulation.

Other prolactin inhibitory factors include somatostatin, acetylcholine, endothelins, gastrin, and growth hormone, while stimulatory factors include thyrotropin-releasing hormone (TRH) (as seen in primary hypothyroidism), angiotensin II, vasopressin, oxytocin, VIP, galanin, and estrogen.

Experiments conducted on rats to elucidate the relationship between the adrenergic system and the regulation of prolactin secretion have focused on stimulating or inhibiting α and β adrenergic receptors. Functional hyperprolactinemia is a complex hormonal interplay of stress-induced neuroendocrine changes involving the dopamine, serotonin and adrenergic systems (36,37). Evidence suggests that the mediobasal hypothalamus and preoptic-anterior hypothalamus harbor the primary adrenoreceptors (38,39). Injecting the α2 agonist clonidine into the mediobasal hypothalamus resulted in a dose-dependent increase in prolactin secretion. This effect was counteracted by the blockade of idazoxan (α2 antagonist). Similarly, the stimulation of prolactin release was induced by isoprenaline (β agonist) and notably attenuated by the β antagonist propranolol. The β2 agonist salbutamol also exhibited efficacy in stimulating prolactin secretion. Conversely, adrenergic agonists such as noradrenaline (mixed α and β), phenylephrine (α1), and tyramine (sympathomimetic) in the mediobasal hypothalamus, failed to elicit an effect on prolactin secretion.

Within the preoptic anterior hypothalamus, noradrenaline and adrenaline were found to stimulate prolactin secretion (40). However, the administration of the α1 agonist phenylephrine failed to stimulate prolactin, indirectly suggesting that the stimulatory effect of noradrenaline in the preoptic anterior hypothalamus is likely due to its action at α2 sites. α2 agonism has been shown to reduce the function of tuberoinfundibular dopaminergic neurons leading to increase prolactin production and secretion (41). Consequently, it was inferred that the activation of α2 and β adrenoceptors in the mediobasal hypothalamus and α2 adrenoceptors in the preoptic-anterior hypothalamus, proximal to prolactin-regulating neurons, leads to heightened prolactin secretion, while the action of α1 in the mediobasal hypothalamus may be inhibitory (42).

Cholinergic activation may have opposite roles in rodents and humans. Cholinergic agonists suppress prolactin release induced by morphine in rats, suggesting that the central cholinergic system has an inhibitory effect on the prolactin release triggered by morphine or β-endorphine, but this cholinergic inhibition does not occur through catecholaminergic neurons (43). Conversely, in humans, cholinomimetic drugs can increase prolactin levels associated with raised plasma β-endorphin, suggesting a stimulatory interplay of cholinergic factors and endogenous opioids on prolactin levels (44), although circulating opioids may not directly relate to central levels.

TIDA neurons express estradiol and progesterone receptors. Estradiol action leads to reduced secretion of dopamine into the portal blood system and mediates a prolactin surge. Progesterone, in addition, suppresses dopamine release being responsible for the plateau phase of the surge (23). Estrogen specifically affects prolactin synthesis by influencing lactotroph cell sensitivity, expression of pituitary dopamine receptor downregulation, and the expression of the prolactin receptor gene (2,34). Ghrelin, a hormone involved in metabolic balance, directly stimulates prolactin secretion at the pituitary level (45).

Tachykinins (substance P, neurokinins A, and B, neuropeptide K, neuropeptide ϒ) can act directly on the lactotroph cell and indirectly within the hypothalamus or posterior pituitary. They have a multifaceted impact on prolactin secretion, with both stimulatory and inhibitory effects. They can stimulate prolactin secretion by stimulating and potentiating the release of oxytocin, vasopressin, TRH, VIP, serotonin and glutamate, and by inhibiting GABA. Tachykinins through paracrine actions can directly increase prolactin within the anterior pituitary. They can also increase dopamine but the overall effect is prolactin elevation. Under specific circumstances, the stimulation of dopamine release can be prominent leading to a decrease in prolactin (46). Endogenous opioids are involved in regulating prolactin secretion, particularly during stressful situations, by reducing the activity of tuberoinfundibular dopaminergic neurons mediated by μ-, κ‑, and δ- opioid receptors, resulting in increased prolactin release (47). Prolonged nicotine exposure has been associated with desensitization of dopamine receptors, diminished dopamine turnover, and a decrease in their abundance within the nigrostriatal pathways (48). These alterations have been suggested to contribute to a diminished prolactin response to opiate blockade observed in individuals who smoke. Similar to opioids, histamine has been shown to induce prolactin production predominantly through inhibiting dopaminergic and stimulating serotoninergic and vasopressin-ergic neurons (49).

Serotoninergic pathways originating from the dorsal raphe nucleus play a physiological role in mediating nocturnal surges and suckling-induced prolactin rises through a serotonin interaction via serotonin type 1 and 2 receptors (5-hydroxytryptamine receptors, 5HT1, and 5HT2). 5-HT could either release a PRL-releasing factor or inhibit dopamine release. The paraventricular nucleus, where serotoninergic pathways terminate, contains postsynaptic serotonin 5-HT1A, 5-HT2, and 5-HT2C receptor subtypes, and possibly 5-HT3 receptors (50). It was shown that the prolactin-releasing effect of serotonin probably occurs mostly via 5-HT1C / 2 receptors because ritanserin (an elective 5-HT1C / 2 receptor antagonist) opposed this effect (51). Serotonin stimulation of prolactin–releasing factor (PRF) neurons in the paraventricular nucleus leads to PRF release (like VIP and oxytocin) mediating hyperprolactinemia. Moreover, serotoninergic stimulation of GABAergic neurons in the tuberoinfundibular-GABA system has been shown to inhibit TIDA cells which contain 5-HT1A receptors, therefore inhibiting dopamine synthesis/release resulting in increased prolactin secretion (52).

Oxytocin, through the posterior pituitary and vasoactive intestinal peptide (VIP) in the anterior pituitary, play significant roles in enhancing PRL gene transcription and modulating dopamine inhibition. Animal studies suggest a potential mediation of VIP by oxytocin to stimulate prolactin secretion (53).

The extensive hormonal regulation of prolactin renders it susceptible to various disturbances caused by different classes of medications.

CLINICAL CHARACTERISTICS

Persistent hyperprolactinemia is associated with disturbances of the gonadal axis leading to interruptions of gonadotrophin-releasing hormone pulsatility and inhibition of luteinizing hormone and follicle-stimulating hormone release (54). Clinical manifestations attributed to hyperprolactinemia predominantly stem from the suppression of the gonadal axis. In premenopausal women, a spectrum of menstrual cycle dysfunctions is observed, spanning from luteal phase shortening to complete amenorrhea, often correlating with elevated prolactin levels. Secondary amenorrhea can be due to hyperprolactinemia in up 30% of patients, and up to 75% of patients with amenorrhea and galactorrhea (55). Beyond these effects, an array of hypoestrogenic indicators may manifest, including symptoms like vaginal dryness, diminished libido, and decreased energy levels. Galactorrhea can be present in up to 80% of females (55,56). In men, the impact of hyperprolactinemia is manifested through a decrease in libido, ranging from diminished sexual desire to oligospermia or even azoospermia attributed to hypogonadotropic hypogonadism. Notably, erectile dysfunction may arise, primarily attributed to the direct inhibitory influence of dopamine, and can be potentially reversed through the administration of dopamine agonists (57). Gynecomastia, on the contrary, is a manifestation of secondary hypogonadism rather than elevated prolactin levels, whereas galactorrhea is rare in men (21).

In both genders infertility can be observed, with diminished bone mineral density. In females, bone mineral density is significantly decreased in women with amenorrhea and increases during treatment and menstrual cycle restoration (58). Additionally, in cases where hyperprolactinemia is attributed to a mass, accompanying clinical indications may encompass headaches, visual field disturbances, cranial nerve palsies, and hypopituitarism. Notably, these manifestations may be the only clinical features in post-menopausal women (21,59).

PSYCHOTROPIC MEDICATIONS

Anti-psychotics and neuroleptic-like drugs are psychotropic medications which primarily exert their anti-psychotic effects through the blockade of DRD2 and D4 receptors in the mesolimbic area. Newer classes of anti-psychotics block 5HT2 and sometimes noradrenergic α1 or α2 receptors (4,35,60). Blockade of D2 receptors in the hypothalamic tuberoinfundibular system and lactotroph cells results in disinhibition of prolactin secretion leading to hyperprolactinemia, being the most common drugs known to induce hyperprolactinemia (61) (Figure 2). On the contrary, strong binding to D2 receptors can extend the half-life of dopamine by approximately 50%. This effect is achieved through two primary mechanisms: direct blockade of the dopamine transporter (DAT) and antagonism of D2 autoreceptors. These processes collectively result in reduced reuptake of dopamine, prolonging its presence in the synaptic area and further stimulating an upregulation of receptors. However, it is important to note that chronic use of anti-psychotics can lead to the reversal of upregulation of DAT (mRNA and protein), potentially contributing to treatment resistance and potentially lower prolactin elevations in the long term. Nonetheless, it is worth mentioning that anti-psychotics typically exhibit a lower affinity for dopamine transporter blockade compared to selective DAT blockers such as nomifensine (62).

Figure 2. Mechanisms of drug-induced hyperprolactinemia with selected examples (adopted from La Torre et al. (2). In addition to opiates, cholinomimetics, PPIs and smoking indirectly also stimulate the opioid receptors. PPIs, Protein pump inhibitors; TCAs, tricyclic anti-depressants; MAO, monoamine oxidase; SSRIs, selective serotonin reuptake inhibitors; SNRIs, serotonin-noradrenaline reuptake inhibitors.

The potency of anti-psychotics and neuroleptic-like medications to induce a rise in prolactin levels varies (Table 1). The level of prolactin increase depends on the anti-psychotic drug (different affinity and selectivity for dopamine receptors; blood-brain barrier penetrating capability, degree of serotoninergic inhibition), the dose administered, and the patient's age and sex (34,35). Lastly, polymorphisms in genes related to dopamine receptors (such as DRD1, DRD2, DRD3) (63), dopamine transporters (SLC6A3), and dopamine-metabolizing enzymes (such as monoamine oxidase and catechol-O-methyltransferase) have been associated with individual variations in response to anti-psychotic treatment and the development of side effects, including hyperprolactinemia (64).

Although direct evidence establishing the involvement of adrenergic receptors in hyperprolactinemia caused by antipsychotic and antidepressant medications remains unproven, indirect indications, as elucidated in Figure 1, suggest the potential implication of these receptors. It is plausible that adrenergic receptors might play a partial role in the hyperprolactinemia induced by these medications.

Table 1. Medications and Their Ability to Cause Hyperprolactinemia
Cluster Name	Subclass mechanism of action		Medications		Prolactin increment	Frequency of prolactin increment (61,65)
Anti-psychotics
First generation anti-psychotics	Antagonize/block dopamine receptors, especially D2 receptors. Can block α1 adrenergic receptors.		Butaperazine		UP to 2-3-fold normal range with dozes 60 mg/daily. Higher in women (66)	High
			Chlorpromazine		Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment) (67)	Moderate/ High
			Flupenthixol		Up to 2-3-fold during the first month, and normalization in the next few months (68)	High
			Fluphenazine		Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment (67). Up to 40-foldof the upper end of the normal range (69)	High
			Haloperidol		Up to 9-fold at the beginning of treatment (3-fold in long-term treatment) (70)	High
			Loxapine		Up to 3-foldof the upper end of the normal range in women (66)	Moderate
			Perphenazine		Up to 40-fold of the upper end of the normal range (69)	Moderate
			Pimozide		?	Moderate
			Prochlorperazine		?	?
			Promazine		Up to 4-fold of the upper end of the normal range (69)
			Thiordiazine		Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment) (67)	High
			Thiothixene		Up to 3-fold with initiation of treatment, up to 2-fold in long-term treatment (71)	Moderate/ High
			Trifluoperazine		?	Moderate
			Veralipride		Up to 10 time increment, transient (72)	High
			Zuclopenthixole		?	?
Second generation anti-psychotics	Dopamine receptors blockade especially D2 receptors, serotonin (5-HT) receptor blockade, glutamate modulation, can antagonize α1 or α2 adrenergic receptors and histamine receptors.		Amisulpiride		Up to 10-fold at the beginning of treatment and remained elevated during treatment but lower levels (68)	Case reports
			Aripiprazole		Reduce prolactin levels (73)	Case reports / No effect/ Reduced prolactin
			Asenapine		Up to 2-fold increment and rarely with higher doses up to 4-fold (35)	Low, Moderate
			Brexpiprazole		Mild increment (74)	Low
			Clozapine		Mild (up to 2-fold) and transient (75)	Case reports or No effect
			Iloperidone		Mild increment, transient (76)	Case reports or No effect
			Levosulpiride		Up to 15-fold normal range (77)	Case reports / Moderate for galactorrhea (78)
			Lurasidone		Up to 10-fold normal range (79)/ no effect (80)	Case reports or No effect
			Molindone		?	Moderate
			Olanzapine		Mild (up to 2-fold) and transient (75)	Low
			Paliperidone		2-10-fold for depot formulations (81)	High
			Perospirone		None (82)	None/ Case reports
			Quetiapine		Mild and transient (75)	Low
			Risperidone		2-10-fold	High (83)
			Sertindole		Mild and transient (75)	?
			Sulpiride		Up to 6-7-fold from baseline, dose dependent effect (84)	High
			Thiethylperazine		?	?
			Ziprasidone		Up to 4-fold from baseline and transient (35,75)	Low
Neuroleptic-like medications
		Block D2 receptors		Domperidone	Up to 10-fold (85,86)	High
				Droperidol	Significant increment after 10 minutes of administration, with peak at 20 minutes (87)	?
				Metoclopramide	Up to 15-fold (2)	High
Anti-depressants
TCAs		Block the reuptake of both serotonin and noradrenaline.		Amitriptyline	2-fold increment on dosage 200/300mg (88)	Low
				Amoxapine	3,5-fold to baseline (89)	High
				Clomipramine	Up to 3-fold increment from baseline (90)	High
				Desipramine	Just above the normal limit with 100 mg oral administration (91)	Low, Controversial
				Imipramine	Up to 4-fold normal range (69)	Controversial
				Nortriptyline	2-fold in the first 2 weeks in one patient (88)	None or Low
SSRI		Block the reuptake of serotonin.		Citalopram/ Escitalopram	Up to 3-fold increment (52)	None or Low (rare reports), Controversial data
				Fluoxetine
				Fluvoxamine
				Paroxetine
				Sertraline
SNRI		Block the reuptake of both serotonin and noradrenaline.		Duloxetine	Up to 2-fold normal range (92)	Case reports
				Milnacipran	Not increased risk of hyperprolactinemia (93)	None
				Venlafaxine	Up to 2-fold normal range, dose related (94)	Case reports
MAO inhibitors		Inhibit the enzyme. Monoamine oxidase, which breaks down serotonin, noradrenaline, and dopamine, though increasing their levels.		Clorgyline	Up to 2-fold from baseline (95)	Low
				Pargyline	Up to 3-fold from baseline (95)	Low
				Phenelzine	Unclear elevation, galactorrhea (96)	Low/ Case reports
Atypical anti-depressants		Inhibit noradrenaline and dopamine reuptake.		Bupropion	No significant change (80)	Case reports
Atypical anti-depressants		Increases the release of both serotonin and noradrenaline.		Mirtazapine	No significant change (80)	Case reports
Serotonin modulators		Modulate serotonin receptors in the brain to enhance serotonin transmission.		Indoramine	(97)	Case report
				Nefazodone	Mild increment from baseline only at acute administration (98)	None/ Case reports
				Trazodone	Up to1.5-fold from baseline (99)	None, Low
				Vortioxetine	Up to 2-fold elevation (100)	Case reports
Selective noradrenaline reuptake inhibitor		Inhibit reuptake of norephinephrine.		Reboxetine	Up to 2-fold from baseline (101)	Case reports
NMDA receptor antagonist		Block NMDA receptors though influencing glutamate neurotransmission.		Esketamine	?	None
Gastric acid reducers
H2 receptor antagonists		H2 receptor antagonists.		Cimetidine	Up to 3-fold after 400 mg IV infusion (102)	Low
H2 receptor antagonists		H2 receptor antagonists.		Ranitidine	Mild increment only in high IV doses (103)	Low
Protein pump inhibitors (PPIs)		Inhibit the activity of the proton pump (H+/K+ ATPase) in the stomach's parietal cells.		Esomeprazole	?	Case reports or No effect
				Lansoprazole	4-fold increment from baseline (104)
				Omeprazole	No significant change (105)
				Pantoprazole	No significant change (106)
				Rabeprazole	No significant change (107)
Opioids
		They activate opioid receptors. Main types of opioid receptors: mu (μ), delta (δ), and kappa (κ).		Apomorphine	By acting as dopamine agonist it lowers prolactin (108)	None
				Heroin	Elevated in addiction (within normal range) compared to healthy control or during abstinence (109)	Moderate in addicted patients that have values over 25 ng/mL
				Methadone	Mild increment, transient increases for several hours following the administration (110)	?
				Morphine	Up to 2-fold increment from baseline (111)	High
Antihypertensives
		It decreases the release of noradrenaline.		Methyldopa	3-4-fold (65) up to 40-fold normal range (69)	Moderate
		Inhibit the storage of neurotransmitters like noradrenaline and serotonin in nerve cells, though decreasing their release.		Reserpine	2.5-fold increment from baseline (112) Up to 40-fold normal range (69)	High
		Block calcium channels in cardiac and smooth muscle cells.		Verapamil	2-fold (113)	Low
Estrogens
		By using as contraceptives they suppress sexual axis.		Estradiol infusion	3-4-fold, dose-dependent, way of administration is important (oral and IV) (114)	Low
				Estradiol withdrawal	?
Gonadotropins and GNRH agonists
		Same as endogenous components, used for fertility induction.		hCG	Up to 4-fold increment, transient (115)	High
				hMG	Up to 2.7-fold increment from baseline, transient (116)	High, Transient
		GnRH agonist.		Leuprolide acetate	1.5-fold higher prolactin in compared to hMG alone, transient (117)	High
Other drugs
Benzodiazepines		Enhances the effects of GABA in the brain.		Diazepam	Mild, dose-dependent (118)	Controversial
Anxiolytics		Serotonin receptor agonist.		Buspirone	2-fold (119)	Case report or No effect
Anxiolytics		α-2 adrenergic agonist.		Clonidine	?	Case reports
Anticonvulsant		Block sodium channels in nerve cells.		Carbamazepine	Less than 2-fold in sleep entrained (120,121)	?
		Block sodium channels in nerve cells.		Phenytoin	Controversial, it can also lower prolactin levels (122)	?
		Enhances the effects of GABA in the brain.		Phenobarbital	Controversial (123)
		Enhances the effects of GABA in the brain.		Valproic Acid	Controversial, it can also lower prolactin (124)	Case reports
Mood stabilizer		Decrease dopamine release and glutamate, increase GABA inhibition.		Lithium Carbonate	Controversial, no effect (183)	None
Antimigraine medication		Calcium channel blocker.		Flunarizine	Mild increment, up to 1.5-fold from baseline (125)	Case reports
Weight loss medications		Increase the release of serotonin and inhibit its reuptake.		Fenfluramine	Mild increment within normal range in previews non-hyperprolactinemic patients (126)	High
Weight loss medications		Inhibit the reuptake of serotonin, noradrenaline, and dopamine.		Sibutramine	4-fold (2,127)	Case report
Anticholinesterase inhibitors		Reversible acetylcholinesterase inhibitor.		Physostigmine salicylate	Less than 100 ng/mL (44).	Low
Prokinetic medication		Stimulate serotonin receptors in the gut.		Cisapride	High increment (up to 200 ng/dL) but in co-administration of other drug inducing hyperprolactinemia (128)	Case reports
Antihistaminic with sedative and antiemetic properties		Block histamine receptors.		Promethazine	?	?
Central Nervous System Stimulants		Increase the release and reduce the reuptake of noradrenaline and dopamine in the brain.		Amphetamine	Mild, only during withdrawal (129)	?
Central Nervous System Stimulants				Methylphenidate	No effect (130)	Case reports/ No effect
ADHD medication		α -2 adrenergic agonist.		Guanfascine	Controversial, it can also lower prolactin (131)	Case reports
Decongestant		Sympathomimetic amine, predominantly α-1 agonist		Pseudoephedrine	Lower prolactin levels (132)	Case reports
Rheumatoid arthritis medications		Reduce inflammation, modify immune response.		Bucillamine	Mild increment within normal range (133)	Case report
Rheumatoid arthritis medications		Reduce inflammation, modify immune response.		Penicillamine	?	Case reports
Osteoporosis medication		Monoclonal antibody that inhibit the receptor activator of nuclear factor kappa-B ligand (RANKL).		Denosumab	?	Case reports
Substance of abuse		Blocks the reuptake of noradrenaline, dopamine, and serotonin in the brain.		Cocaine	Decrease prolactin levels (134) Mild increment only during withdrawal (129)	Case reports
		Increases the release and inhibits the reuptake of serotonin and to some extent, dopamine and noradrenaline.		Ecstasy	Mild or no effect (135)	?
		Stimulates nicotinic acetylcholine receptors, leading to the release of neurotransmitters like dopamine and noradrenaline.		Smoking	Mild increment, transient (136)	Moderate
Anti-HIV medications		Protease inhibitors that prevent the cleavage of viral proteins and thereby inhibiting viral replication.		Ritonavir / Saquinavir	Mild (137)	Case reports
Radiotherapy		Use of high-energy radiation to damage the DNA within the targeted cells.		Intracranial radiotherapy	?	Moderate (138)

Frequency of increase to abnormal prolactin levels with chronic use: high: >50%; moderate: 25 to 50%; low: <25%; none or low: case reports. The effect may be dose-dependent. Drugs marked with blue have controversial data or decrease prolactin levels as explained in the table. Where we could not identify reliable data for the parameters in the table we added a question mark. *First-generation anti-psychotics, non-selective dopamine receptors antagonists. **Second-generation anti-psychotics.

Anti-Psychotics

Anti-psychotics are traditionally classified as first- and second-generation, but more recently a new classification taxonomy has been developed by McCutcheon et al. to express different receptor affinity of different anti-psychotics. Due to the impossibility to include in this new classification all drugs that cause hyperprolactinemia, we have used the old classification (Table 1) (139,140).

The first-generation anti-psychotics are typically associated with more severe hyperprolactinemia (2-3-fold increment), whereas second-generation drugs have lower D2 affinity and stronger blockade of 5HT2A receptors leading to milder prolactin elevations (1-2-fold), except risperidone, paliperidone, and amisulpiride. Amisulpiride has the greatest potential to cause hyperprolactinemia of all anti-psychotics (4).

The first-generation anti-psychotics, such as fluphenazine and haloperidol, act as non-selective dopamine receptors antagonists (2,10). The therapeutic effects on psychotic symptoms occur through D2 and D4 receptor binding in the mesolimbic area, while side effects are mediated by D2 blockade in the striatal area (linked to extrapyramidal effects) and in the hypothalamic infundibular system (linked to hyperprolactinemia) in more than 50% of patients. A clinical trial involving 69 patients examined the effects of various anti-psychotic medications on prolactin levels, including chlorpromazine, depot haloperidol, fluphenazine, zuclopenthixol, sulpiride, pimozide, droperidol, and flupenthixol. The study found a significant elevation in prolactin levels only in females, with a mean level of 1106 mIU/L (52 ng/mL) compared to the normal range of <480 mIU/L (22.6 ng/mL). In males, the mean prolactin levels were within the normal range, which may be attributed to the significantly lower total daily dose of chlorpromazine used in males (199.0-220.1 mg/day) compared to females (384.4-302.48 mg/day, P<0.05) (7).

Second-generation anti-psychotics with lower D2 affinity led to milder prolactin elevations (1-2-fold), except for paliperidone, risperidone, and amisulpiride whose effect on prolactin is similar to the first-generation neuroleptics. Chlorpromazine, loxapine, olanzapine and quetiapine have variable effects on prolactin secretion, while aripiprazole, clozapine, iloperidone, lurasidone have little or no effect on prolactin secretion (35).

An important factor contributing to variations in the induction of hyperprolactinemia by different anti-psychotic medications is the blood-brain barrier. Permeability glycoprotein transporter (P-gp), coded by the ABCB1 gene, is expressed in various tissues including in the cells of the blood-brain barrier. P-gp plays a role in actively transporting hydrophobic drugs with a molecular weight greater than 400 Da out of the brain, thus protecting the brain from these medications; therefore, this protein can change drug bioavailability (141,142).

The affinity of risperidone, paliperidone, and amisulpiride (prolactin rises up to 10-fold with these drugs) for P-gp is approximately twice that of olanzapine and chlorpromazine (prolactin rise is up to 3-fold with these drugs), and four times greater than haloperidol and clozapine (prolactin rise can be high initially but usually reduces with time) (143). The higher affinity of risperidone, paliperidone, and amisulpiride to P-gp could, among other mechanisms, partly explain the greater induction of hyperprolactinemia by these drugs, as P-gp does not allow them to enter the brain via the blood-brain barrier. Therefore, the portal circulation of the anterior pituitary delivers a somewhat higher concentration of these drugs to the lactotrophs, which are located outside the blood-brain barrier, to inhibit the D2 receptors (144).

Aripiprazole can act as a partial agonist at D2 receptors and display partial agonist activity at 5HT1A receptors, while also acting as an antagonist at 5HT2A receptors. Antagonism at these receptors can help to normalize prolactin levels since 5HT2A receptor activation has been associated with increased prolactin release. That is why it is considered a prolactin secretion modulator (145). Its role in prolactin levels has been investigated in a study involving both retrospective and prospective components (146). The retrospective part of the study included 30 patients undergoing risperidone treatment, when it was observed that after 6 months of treatment, prolactin levels remained high although somewhat lower than at the start of observation. In the prospective part of the study, 30 other patients were divided into two groups: one group receiving risperidone alone at a daily dosage of 2-4 mg and the other group receiving a combination of risperidone and aripiprazole at a daily dosage of 5-10 mg. The group receiving adjunctive aripiprazole exhibited significantly lower serum prolactin levels compared to the risperidone-only group at weeks1 (914±743 vs 1567±1009 mU/L), 2 (750±705 vs 1317±836 mUI/L) and 6 (658±590 vs 1557±882 mUI/L). Notably, during aripiprazole treatment, prolactin levels at weeks 1, 2, and 6 were significantly lower than at baseline (P< 0.05) (at baseline patients were treated with risperidone as monotherapy), suggesting that aripiprazole may effectively alleviate risperidone-induced hyperprolactinemia. Similar findings supporting the role of aripiprazole in reducing prolactin levels have been reported in other studies (147). Combination therapy presents a promising therapeutic approach for adjunctive treatment or for transitioning from risperidone to mitigate hyperprolactinemia (146).

More recently, a new medication SEP-363856, a trace amine-associated receptor 1 (TAAR1) and 5HT1A agonist, has been developed to treat schizophrenia. Its mechanism of action is not based on D2 antagonism, and has a favorable effectiveness and tolerability profile, without causing hyperprolactinemia (148). This category of medication serves as compelling evidence for the significant involvement of dopamine receptors in drug-induced hyperprolactinemia, and it is a future viable therapeutic choice for patients experiencing adverse effects associated with hyperprolactinemia.

DRUG-INDUCED HYPERPROLACTINEMIA IN PEDIATRIC PATIENTS

Anti-psychotic medications have been found to induce hyperprolactinemia in the pediatric population as well as in adults. In a trial involving 35 children and adolescents with early-onset psychosis, primarily diagnosed with childhood-onset schizophrenia or psychotic disorder not otherwise specified, prolactin levels were measured after a 3-week washout period, as well as after 6 weeks of treatment with haloperidol, olanzapine, and clozapine (149). Following the 6-week treatment period, haloperidol (9 of 10 patients – mean age 13.4 years) and olanzapine (7 of 10 patients– mean age 15.9 years) resulted in prolactin levels above the upper limit of normal. The mean increase was 5.2-fold for haloperidol and 2.4-fold for olanzapine. The prolactin response did not show statistically significant differences between females and males treated with haloperidol and olanzapine. Clozapine (22 patients, mean age 14.7) caused a small but significant rise in females (1.2-fold) but levels remained in the normal range for all patients. There was no rise in males. Why this difference between females and males is only on clozapine remains unclear and difficult to explain. However, in a study involving 36 girls aged 8-17 years, mean prolactin levels were higher in girls compared to boys, with the most significant increase occurring around the age of 13y, correlating with menarche. A highly significant correlation was found between increases in plasma prolactin and estradiol levels between the ages of 11 and 13 years. Girls with long menstrual cycles (>28 days) between the ages of 14 and 16 years had higher prolactin levels (p<0.05) (150). Even though the mean age of patients on clozapine was above 13 years, we do not possess information on the duration of the menstrual cycle of those girls, as if it is longer, the physiologic estrogenization because of the longer menstrual cycle can impact the range of prolactin elevation. In any case, the population sample size was relatively small to draw definitive conclusions and to provide answers why this happens only in clozapine patients (149).The authors concluded that the prolactin response in male children and adolescents treated with haloperidol or olanzapine was significantly higher than that observed in adult males. However, the prolactin response in female children and adolescents after haloperidol treatment did not differ significantly from that of adult females in similar studies, possibly due to the adult similarity of estrogen status seen in female adolescents (149). Aripiprazole in another study showed a lesser prolactin increase than olanzapine, quetiapine, and risperidone, similar to the adult population (151).

In another clinical trial involving 396 children and adolescents (aged 14.0 ± 3.1 years), the impact of anti-psychotic medications on prolactin levels was studied. The medications involved risperidone, olanzapine, quetiapine, and aripiprazole. Risperidone caused the highest incidence of hyperprolactinemia (93.5%) and had the highest peak prolactin levels (median = 56.1 ng/mL) followed in order by olanzapine, quetiapine, and aripiprazole. Menstrual disturbances were the most prevalent side effect (28.0%), particularly with risperidone (35.4%). Notably, severe hyperprolactinemia was associated with decreased libido, erectile dysfunction, and galactorrhea (152).

In conclusion, a comprehensive meta-analysis comprising 32 randomized controlled trials with a total of 4643 participants, with an average age of 13 years, has demonstrated that risperidone, paliperidone, and olanzapine are associated with a significant increase in prolactin levels among children and adolescents. Conversely, aripiprazole is linked to a notable decrease in prolactin levels in this age group. It is worth noting that haloperidol was not included in these studies, resulting in an absence of evidence regarding its prolactin-related effects in this population (153).

These findings underscore that haloperidol, risperidone, paliperidone and olanzapine are potent inducers of hyperprolactinemia in children and adolescents, mirroring observations in the adult population. A comprehensive listing of medications associated with hyperprolactinemia in children can be found in Table2.

Table 2. Drugs Reported to Induce Hyperprolactinemia in Children and Adolescents
Medication class	High >50 percent of patients	Moderate 25-50 percent of patients	Low <25 percent of patients	Case reports
Anti-psychotics, first-generation 'typical'	Fluphenazine (154) Haloperidol (149,155)	Chlorpromazine (156) Loxapine (157) Pimozide (158,159)
Anti-psychotics, second-generation 'atypical'	Paliperidone (160,161) Risperidone (152,155,162–164)	Asenapine (165) Molindone (166) Olanzapine (149,152) Lurasidone (167,168)	Ziprasidone (169) Quetiapine (152,162)	Clozapine (149) Aripiprazole* (152,170) Amisulpride (171) Brexpiprazole (172)
Anti-depressants	Clomipramine (173)		Desipramine (174)	Bupropion (175) Citalopram (176) Escitalopram (177) Fluoxetine (178) Sertraline (179) Duloxetine (177) Paroxetine (180) Venlafaxine (181)
Anti-emetics and gastrointestinal medications	Metoclopramide (182–184) Domperidone (185,186)			Omeprazole (187) Lansoprazole (187) Cisapride
Others	Fenfluramine (188)		Estrogens (189) Triptorelin (190)	Clonidine (191) Methylphenidate (181) Guanfacine (181) Valproic acid (181) Penicillamine (181)

Table 2. Drugs Reported to Induce Hyperprolactinemia in Children and Adolescents

Medication class

High

>50 percent of patients

Moderate

25-50 percent of patients

Low

<25 percent of patients

Case reports

Anti-psychotics, first-generation 'typical'

Fluphenazine (154)

Haloperidol (149,155)

Chlorpromazine (156)

Loxapine (157)

Pimozide (158,159)

Anti-psychotics, second-generation 'atypical'

Paliperidone (160,161)

Risperidone (152,155,162–164)

Asenapine (165)

Molindone (166)

Olanzapine (149,152)

Lurasidone (167,168)

Ziprasidone (169)

Quetiapine (152,162)

Clozapine (149)

Aripiprazole* (152,170)

Amisulpride (171)

Brexpiprazole (172)

Anti-depressants

Clomipramine (173)

Desipramine (174)

Bupropion (175)

Citalopram (176)

Escitalopram (177)

Fluoxetine (178)

Sertraline (179)

Duloxetine (177)

Paroxetine (180)

Venlafaxine (181)

Anti-emetics and gastrointestinal medications

Metoclopramide (182–184)

Domperidone (185,186)

Omeprazole (187)

Lansoprazole (187)

Cisapride

Others

Fenfluramine (188)

Estrogens (189)

Triptorelin (190)

Clonidine (191)

Methylphenidate (181)

Guanfacine (181)

Valproic acid (181)

Penicillamine (181)

*Aripiprazole is a partial agonist at the type 2 dopamine receptor and display partial agonist activity at the type 1A serotonin receptor (5HT1A) and antagonist at 5HT2A receptor. It can be used in combination with other psychotropic medications to reduce prolactin levels. Aripiprazole itself can sometimes cause mild hyperprolactinemia (192).

CLINICAL MANAGEMENT OF ANTI-PSYCHOTIC-INDUCED HYPERPROLACTINEMIA

Drug-induced hyperprolactinemia should be considered in the differential diagnosis of elevations of prolactin levels, sometimes greater than 200 µg/L (4260 mIU/L). Particularly when serum prolactin levels exceed 80-100 µg/L (1700-2130 mIU/L), pituitary magnetic resonance imaging (MRI) should be performed to rule out the presence of any underlying pituitary or hypothalamic masses that may contribute to hyperprolactinemia (193). According to the guidelines from the Endocrine Society, in symptomatic patients suspected of having drug-induced hyperprolactinemia, it is recommended the first test to diagnose drug-induced hyperprolactinemia is the discontinuation of the medication for 3 days or switch to an alternative drug (e.g. a prolactin-sparing anti-psychotic (e.g. aripiprazole), or an anti-psychotic with lower dopamine antagonist potency (Table 1) followed by retesting of serum prolactin levels.

However, any discontinuation or substitution of anti-psychotic agents should be done in consultation with the patient's psychiatric physician. If discontinuation is not possible or if the onset of hyperprolactinemia does not coincide with therapy initiation, obtaining a pituitary MRI is recommended (despite prolactin levels) to differentiate between medication-induced hyperprolactinemia and hyperprolactinemia caused by a pituitary or hypothalamic mass (194).

Before initiating treatment with an anti-psychotic medication, it is advised that clinicians inquire about the patient's previous treatment experience, sexual dysfunction, menstrual history (including irregularities and menopausal status), as well as any history of galactorrhea. Additionally, obtaining a baseline prolactin level is recommended before starting treatment (195). This pre-treatment screening for hyperprolactinemia can help determine whether subsequently elevated prolactin levels are due to medication-induced factors (196) and make the diagnosis easier without the need to perform further imaging.

While treatment of hyperprolactinemia in patients receiving anti-psychotics may not always be necessary, in cases where clinical hypogonadism is evident, several options are available (193). The initial step is to discontinue the drug if clinically feasible. If discontinuation is not possible, switching to a similar anti-psychotic that does not cause hyperprolactinemia is suggested. If neither of these options is feasible, cautious administration of a dopamine agonist may be considered in consultation with the patient's physician (194). It is worth noting that these interventions only result in the normalization of prolactin levels in approximately half of the patients receiving anti-psychotics, and careful psychiatric monitoring is required due to the possibility of psychosis exacerbation with dopamine agonists (193).

For patients with long-term hypogonadism demonstrated by hypogonadal symptoms or low bone mass, the use of estrogen or testosterone replacement is recommended. In rare instances where patients receiving anti-psychotics also present with a pituitary tumor, treatment options primarily revolve around tumor-specific interventions, considering especially optic chiasm compression. When a non-functioning tumor is suspected, the above options to eliminate the drug-induced component of hyperprolactinemia should be considered, with supervised short-term cessation of the medication to clarify drug-induced or tumor-derived hyperprolactinemia (193).

In addition to the interventions mentioned earlier, addressing fertility concerns in patients with drug-induced hyperprolactinemia may require further measures. Normalization of prolactin levels through medication adjustment or dopamine agonist therapy can often lead to the restoration of fertility. In situations where fertility is not spontaneously regained, fertility treatment with gonadotrophins or assisted reproductive procedures may be necessary.

NEUROLEPTIC-LIKE MEDICATIONS

Metoclopramide and domperidone are anti-emetic and gastrointestinal motility agents known also as neuroleptic-like medications which can increase pituitary prolactin secretion and breast milk production by a dopamine antagonistic action (Figure 1).

Metoclopramide is a central and peripheral D2 receptor antagonist (197). Its administration is followed by an acute increase in prolactin levels up to 15-fold above the baseline that persists in chronic administration of the drug (2). Even though the majority of patients treated with metoclopramide develop hyperprolactinemia (Table 1), related symptoms such as amenorrhea, galactorrhea, gynecomastia, and impotence remain unclear (2).

Domperidone is a peripheral D2 antagonist (it does not cross the blood-brain barrier) used for treating intestinal motility disorders, especially for the prevention of gastrointestinal discomfort with dopaminergic treatment in Parkinson's disease (198), as it antagonizes the D2 receptors in the upper gastrointestinal tract. It also reaches the D2 receptors on lactotroph cells inducing hyperprolactinemia, and can be used for stimulating lactation, for example in women with preterm infants, or an adoptive parent (199,200), and even in transgender women who wish to breastfeed (201,202). It can cause a 10-fold elevation in prolactin levels with normalization of prolactin levels after three days (85,86). Neuroleptic-like medications are summarized in Tables 1 and 3.

Table 3. H2 Receptor Antagonists, Opioids, Anti-Hypertensives, PPIs, Estrogens, and Other Drugs and Their Ability to Cause Hyperprolactinemia.
Medication class	High >50% of patients	Moderate 25-50% of patients	Low <25% of patients	Case reports
Anti-emetic and gastrointestinal	Domperidone Metoclopramide		Prochlorperazine	Esomeprazole Omeprazole Lansoprazole Cisapride
H2-receptor antagonists			Cimetidine Ranitidine
Anti-hypertensives		Methyldopa	Verapamil
Others	Fenfluramine Opioids		Estrogens	Protease inhibitors Cocaine Bucillamine Clonidine Methylphenidate Guanfascine Valproic Acid Penicillamine

ANTI-DEPRESSANTS

Anti-depressants can be classified based on their structure and mechanism of action into tricyclic anti-depressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), monoamine oxidase (MAO) inhibitors, atypical anti-depressants, serotonin modulators, selective noradrenaline reuptake inhibitor, and NMDA (N-Methyl-D-Aspartate) receptor antagonists (203). Data on their ability to cause hyperprolactinemia are controversial, as described below. The two main mechanisms, both related to elevated serotoninergic tonus, explain how anti-depressants can induce hyperprolactinemia by indirect modulation of prolactin release by serotonin and serotonin stimulation of GABAergic neurons (50) Adrenergic receptors involvement in their ability to cause hyperprolactinemia remains unclear. (Figure 1).

Data on the incidence of hyperprolactinemia with anti-depressant medications, especially SSRIs, MAO inhibitors, and some TCAs, suggest that they can cause modest and generally asymptomatic hyperprolactinemia (4,193). Their ability to cause hyperprolactinemia is summarized in Table 1.

TCAs

TCAs, such as amitriptyline, desipramine, clomipramine, and amoxapine, can induce sustained mild hyperprolactinemia (2) (Table 1). They manifest their mechanism of action by blocking the reuptake of noradrenaline and serotonin, through increasing serotoninergic tonus, leading to hyperprolactinemia. Amitriptyline's ability to cause hyperprolactinemia seems to be dose-dependent. A dosage of 150-250 mg/day to 5 patients showed no effect on prolactin levels after 3-7 weeks (204), whereas a dosage of 200-300 mg/day caused a 2-fold increment of prolactin levels in two of nine chronic treated patients (88). In 13 patients with depression taking amitriptyline or desipramine, prolactin levels were studied after intravenous injection of tryptophan (serotonin precursor). It was observed that tryptophan-induced prolactin elevation was significantly increased compared with a preceding placebo period (205). A similar tryptophan test was performed with clomipramine 20 mg vs placebo in 6 normal subjects. Levels of prolactin increased after tryptophan infusion in pretreated patients with clomipramine (206),suggesting serotonin involvement in this hyperprolactinemia.

The effect of the traditional TCA imipramine on serum prolactin levels is controversial. A five-week double-blind study on patients with depression taking imipramine did not show any significant change in serum prolactin (207). However, another study in young healthy men showed that imipramine’s effect on prolactin is dose-dependent (usually therapeutic dose is 50-150 mg): oral administration of 100 mg, but not of 40 mg, led to a consistent rise in prolactin levels after 3 hours of administration, but the rise was mild (maximum 25.85 ng/mL (550 mUI/L) (91).

Data regarding nortriptyline's ability to induce sustained hyperprolactinemia are lacking. Anyway, over a 4-6 week treatment of 8 patients with nortriptyline up to 150 mg/daily, no difference was found between placebo and the medication group. In only one patient was observed a transitional 2-fold elevation in the first 2 weeks (88).

Amoxapine, which is an anti-depressant with neuroleptic properties as well, is found to increase prolactin levels in 10 patients approximately 3.5-fold compared to baseline and more than desipramine in 12 patients, where almost no difference with baseline was observed (89). The proposed mechanism of hyperprolactinemia involves the blockade of the D2 receptor in tuberoinfundibular neurons or the anterior pituitary gland (2).

SSRIs

SSRIs enhance serotonin activity via inhibition of neuronal serotonin reuptake. This could be the most prominent mechanism leading to a prolactin elevation. A review of 13 case reports showed prolactin levels between 28 and 60 ng/mL (595 and 1276 mUI/L) (52). In a French study, 27 of 159 cases (17%) had SSRI-induced hyperprolactinemia with sertraline being the most prominent, followed by fluoxetine, paroxetine and fluvoxamine. Only citalopram was found not to increase prolactin levels significantly (208). However, in another study fluoxetine, paroxetine, and fluvoxamine were found again to elevate prolactin levels, but they also found citalopram to induce hyperprolactinemia. In this study duloxetine, milnacipran, and sertraline (which was the most prominent in the previous study) were not associated with an increased risk of hyperprolactinemia (93). Fluoxetine-induced hyperprolactinemia was found in patients with major depression (4.5% of men and 22.2% of women) following 12 weeks of fluoxetine treatment (209).Differences in the ability to cause hyperprolactinemia can be attributed to the variations in the affinity of the SSRIs for dopamine, histamine, and GABA receptors.

The Nurses’ Health Study and its follow-up study assessed anti-depressant use and circulating prolactin levels in 610 women (including 267 anti-depressant users) with two measurements of prolactin an average of 11 years apart (210). In this study, mean prolactin levels were similar among SSRI users (13.2 µg/L, (280 mUI/L), 95% CI 12.2-14.4), users of other classes of anti-depressants (12.7 µg/L (270 mUI/L), 95% CI 11.0-14.6), and non-users (13.1µg/L, (278 mUI/L), 95% CI 12.8-13.4) (210). However, the duration and dosage of anti-depressant use at the time of prolactin sampling had not been assessed, as the participants had only responded as current anti-depressant users/non-users on a questionnaire.

MAO Inhibitors

Monoamine oxidase is an enzyme responsible for breaking down neurotransmitters such as serotonin, noradrenaline, and dopamine in the brain. Inhibition of this enzyme is expected to increase the levels of all those neurotransmitters. Even though increased dopamine is suspected to be related to lower prolactin levels, probably the serotonin increment prevails and that is why this class of medications is related to hyperprolactinemia; or dopamine and serotonin increment neutralize each other and no difference in prolactin levels is seen.

MAO inhibitors with serotoninergic activity (pargyline and cordyline) can cause modest and generally asymptomatic hyperprolactinemia (2,61). Phenelzine was observed to persistently increase prolactin levels in 4 of11 patients, which returned to normal during a placebo week and rose again in all 4 patients after treatment restart (88).

Atypical Anti-Depressants

Mirtazapine's mechanism of action is different from other anti-depressants, as it does not inhibit the reuptake of serotonin or noradrenaline, but it increase the release of serotonin and noradrenaline (211). In any case, prolactin levels did not show any difference in 8 healthy male subjects pre- and post-mirtazapine 15 mg oral administration (212).

Serotonin Modulators

Trazodone acts through dual inhibition of serotonin reuptake and serotonin type 2 receptors, coupled with antagonism of histamine and α-1-adrenergic receptors (213). In 12 patients with depression, 150 mg of trazodone for 3 weeks caused significantly higher prolactin levels after 12 hours, 1 week, and 2 weeks of treatment compared to baseline. Higher levels were after the first week 15,3+/-8,5 ng/mL (325 +/- 180 mUI/L) compared to baseline 9,1 +/- 5,6 ng/mL (194+/-119 mUI/L) (99).

Selective Noradrenaline Reuptake Inhibitor

Reboxetine is a selective noradrenaline reuptake inhibitor that was shown to increase prolactin in healthy men after acute administration, but this effect can be reversed if reboxetine is simultaneously administered with α2-blocker mirtazapine, suggesting a role of α2-receptors in the enhancement of prolactin release after reboxetine (214). In any case, conflicting data are present even with this drug, with one other study showing no difference between pre-treatment and after-treatment prolactin levels in patients taking up to 8 mg reboxetine for 4 weeks in 17 patients (215). This discrepancy can be due to the limited number of patients or other neuroendocrine mechanisms still less explored.

SNRI medications are described to cause only mild and rare elevations on prolactin levels, whereas (94), esketamine (NMDA receptor antagonist)is not described to cause hyperprolactinemia.

Summary

In summary, controversial data are available on anti-depressant-induced hyperprolactinemia. Routine monitoring of prolactin levels in patients taking anti-depressants is not recommended unless symptoms related to prolactin increase (in premenopausal women: menstrual cycle dysfunction leading to amenorrhea, oligomenorrhoea, anovulatory cycles, low libido, and energy; in men: erectile dysfunction, decreased energy and libido, decreased muscle mass, decreased body hair; and for both of them osteopenia, galactorrhea and infertility) occur (50,193). If the prolactin serum level is elevated (> 25 µg/L (531 mUI/L), in these patients a differential diagnosis is needed. The proposed approach is to withdraw the anti-depressant drug slowly over 2 weeks and replace it with another anti-depressant less likely to cause hyperprolactinemia, then reassess symptoms and prolactin levels after 2-4 weeks (193). If the serum prolactin remains elevated, other causes of hyperprolactinemia should be addressed by an endocrinologist. Another approach is to perform a pituitary MRI if the replacement of the anti-depressant is difficult to manage.

GASTRIC ACID REDUCERS

Histamine-Receptor Inhibitors

Histamine, a CNS neurotransmitter, binds to both H1 and H2 receptors. It can stimulate prolactin secretion via H1 receptors by inhibiting the dopaminergic system. On the contrary, histamine can also inhibit prolactin secretion via H2 receptors using a non-dopaminergic mechanism involving β-endorphin, vasoactive intestinal peptide, vasopressin, or TRH (216), all of which act as prolactin-releasing factors (49) (Figure 1).

In the French Pharmacovigilance Study, H2 receptor antagonists were found to contribute to 5% of drug-induced hyperprolactinemia, primarily with ranitidine (odds ratio = 4.43; 95% CI: 1.82–10.8) (11). Other studies have shown that H2-receptor antagonists such as cimetidine and ranitidine can elevate prolactin levels (217,218). Specifically, cimetidine caused a three-fold increase in prolactin levels after a 400 mg IV infusion, although this effect was not observed with oral administration of 800 mg cimetidine in healthy individuals (102) (Table 1,3).

Interestingly, it has been observed that systemic administration of the H2 agonist impromidine does not prevent cimetidine-induced hyperprolactinemia. In contrast, pre-administration of benzodiazepines or GABA lowered the prolactin response. This suggests that cimetidine-induced hyperprolactinemia may be mediated through neurotransmitters in the GABA-ergic system (219).

Proton-Pump Inhibitors

Proton-pump inhibitors (PPIs) have been recently reviewed regarding the risk of hyperprolactinemia and related sexual disorders observed with long-term use (220). The exact mechanism by which PPIs increase prolactin levels is not fully understood; possible explanations include inhibition of dopamine receptors, interference with other dopamine receptors, involvement of the serotoninergic pathway, modulation of the opioid pathway, and a potential role in decreasing prolactin clearance (220) (Figure 2). In addition, PPIs can increase gastrin levels in chronic use especially in females using high doses (221). As gastrin can act as prolactin-inhibitory factor (108), this antagonistic effect may explain the relatively mild hyperprolactinemia occurring with this class of drugs. Esomeprazole has a mild inhibitory effect on CYP3A4, which leads to decreased metabolism of estrogen, thereby increasing serum estrogen levels which can stimulate the production of prolactin (222). Gynecomastia, impotence, irregular menses, and galactorrhea have been described with PPI use. Hyperprolactinemia occurred less often than sexual disorders, and most cases of hyperprolactinemia were reported with omeprazole, esomeprazole, and lansoprazole use (e.g. 4-fold increment with lansoprazole) (104) (Table 1,3). Pantoprazole and rabeprazole were only sporadically associated with hyperprolactinemia. The authors assert that the occurrence of sexual dysfunction in individuals using PPIs, despite having normal prolactin levels, may be attributed to the development of low vitamin B12 levels, hypomagnesaemia and iron deficiency resulting from PPI usage. These nutritional deficiencies have been implicated in the manifestation of sexual disorders in other studies (220,223,224).

OPIOIDS

Endogenous opioids, morphine, and related drugs, activate ε-, μ-, κ- and δ- opioid receptors in the hypothalamus, modulating pituitary hormone secretion. They do not possess direct effects on pituitary cells (225,226). They inhibit the gonadal axis via ε receptors (GnRH suppression) and stimulate prolactin production by reducing the activity of tuberoinfundibular dopaminergic neurons via μ, κ and δ opioid receptors – mostly μ receptors as the μ receptor opioid antagonist naloxone prior to morphine and methadone use prevents opioid-induced hyperprolactinemia (227). Indirect stimulation of prolactin release can be mediated by stimulating prolactin-releasing factors production (the serotoninergic pathways discussed above) (228) (Figure 2). Hyperprolactinemia can then lead to additional gonadal axis suppression. In addition, opioids can modulate the corticotroph axis via κ and δ opioid receptors and the somatotroph axis via μ, κ and δ opioid receptors (226). Additionally, opioids manifest negative effects on bone health through direct inhibition of osteoblasts by opioids, gonadal axis suppression, altered mental status, and other comorbidities (chronic conditions, smoking, alcohol use) (229).

Acute intravenous or intra-ventricular administration of endogenous opioids leads to a rapid plasma prolactin increase in a dose-dependent manner (44,225). Chronic use of opioids effects on prolactin can vary: oral opioids for chronic pain increase prolactin, but morphine administered intrathecally for chronic non-cancer pain had no effect on prolactin (226). Hyperprolactinemia induced by opioids can be symptomatic: painful gynecomastia, galactorrhea, and hypogonadism have been reported in chronic opioid users. These can be alleviated with discontinuation or reduction of opioid dose, and sometimes dopamine agonists such as bromocriptine (226). Methadone induces a transient increase in prolactin levels, whereas chronic methadone users have normal basal prolactin levels (110), (Table 1, 3).

On the contrary, in a study involving six patients with hyperprolactinemia and amenorrhea, the use of naltrexone, an opioid antagonist, was investigated to determine if blocking endogenous opioids could improve the sexual axis. On the first day of naltrexone administration, significant increases were observed in the mean concentration of luteinizing hormone (LH), LH pulse amplitude, and estradiol levels compared to the control day. This indicated a prompt partial reactivation of the hypothalamic-pituitary-gonadal axis as a result of naltrexone, leading to heightened gonadotrophin levels and subsequent release of estradiol. However, it was found that the effect of opioid antagonism did not result in a sustained increase in estradiol secretion with chronic treatment. Additionally, prolactin levels continued to increase over time (mean prolactin level 255 ± 121 microgram/L), despite the initial improvement in the gonadal axis. This study demonstrated that although prolactin-induced suppression of the gonadal axis can be reversed to some extent by acute opioid antagonism, it is not an effective treatment for revitalizing the gonadal axis in the long term. Possible explanations for this lack of sustained effect include desensitization of the hypothalamic-pituitary unit for the effects of opioid receptor blockade and other disruptors of the axis, which may counteract the positive effects of opioid antagonism (230,231).

For endocrinopathies caused by opioids, including hyperprolactinemia, potential management choices include reducing or discontinuing opioid usage whenever feasible and exploring alternative pain relief therapies for chronic pain situations. Hormonal replacement therapy can be considered for hypogonadism and hypoadrenalism (226).

ANTIHYPERTENSIVES

Some antihypertensive medications including α-methyldopa, verapamil, labetalol, and reserpine, have been associated with hyperprolactinemia. This phenomenon is attributed mainly to the potential inhibition of dopaminergic pathways, highlighting the complex interplay between anti-hypertensive therapy and endocrine function. Other mechanisms are drug-specific and will be explained below.

α -methyldopa is an α-adrenergic inhibitor that leads to the suppression of monoamine synthesis, including noradrenaline, dopamine, and serotonin, which likely contributes to its anti-hypertensive effect. It causes hyperprolactinemia through the inhibition of dopamine synthesis by competitive inhibition of DOPA decarboxylase which transforms L-dopa into dopamine (61) (Figure 2). Long-term treatment resulted in elevated basal prolactin levels (3-4-fold), while a single dose of 750-1000mg reaches a peak of high prolactin level after 4-6 hours of administration (232). Gynecomastia is the most common endocrine side effect.

Calcium channel blockers are other drugs studied for their potential to cause hyperprolactinemia. The dihydropyridine class was found to have no effects on prolactin levels. Whereas, from the non-dihydropyridine class, which mainly blocks L-type of calcium channel receptors in the heart, only verapamil was found to cause 2-fold persistent hyperprolactinemia (and galactorrhea),while drug discontinuation reversed hyperprolactinemia in all patients (113). A clinical trial suggested that verapamil acts by reducing dopamine release in the tuberoinfundibular pathway through calcium influx inhibition (Figure 2), possibly by N-calcium channels which are known to be involved in the regulation of dopamine release and other neurotransmitters (233).

Labetalol is an α- and β-adrenoceptor blocker anti-hypertensive that has been reported to increase prolactin levels when administered intravenously, but not when administered orally (100 or 200 mg) as labetalol cross the blood-brain-barrier only in negligible amounts. The increase in prolactin release caused by intravenous labetalol is not readily explained by its interference with adrenergic receptors. The exact mechanism underlying this effect of the drug is currently unclear, but it is possible that labetalol's ability to block dopamine activity (anti-dopaminergic activity) might be involved in this response (234) (Figure 2). Pre-treatment with levodopa and carbidopa can prevent prolactin response after labetalol (235), suggesting dopamine pathway involvement suppression inside the blood-brain-barrier.

Reserpine is a rauwolfia alkaloid previously used for the treatment of hypertension as well as psychosis, schizophrenia, and tardive dyskinesia; it reduces dopamine by inhibiting their hypothalamic storage in secretory granules (236), and by blockade of vesicular monoamine transporter type 2 in monoamine neurons (237), leading to hyperprolactinemia. Prolactin levels are higher during treatment with reserpine than 6 weeks after discontinuation of the drug. Increased incidence of gynecomastia and breast cancer has also been reported among patients on anti-hypertensive therapy with reserpine (236). The ability of anti-hypertensives to cause hyperprolactinemia is summarized in Tables 1 and 3.

ESTROGENS

Estrogens stimulate prolactin secretion by several mechanisms: They bind to specific intracellular lactotroph cells receptors, though enhancing prolactin gene transcription and synthesis (238). They also inhibit tuberoinfundibular dopamine synthesis, stimulate lactotroph cell hyperplasia, downregulate dopamine receptor expression, and modify lactotroph responsiveness to other regulators (23,239) (Figure 2). Estrogen-induced hyperprolactinemia is dependent on the degree of estrogenization. Higher levels of estrogens in pregnancy and during ovulation increase prolactin levels with the last, contributing to a higher normal range of prolactin in pre-menopausal women.

Studies documenting the incidence of hyperprolactinemia showed that women on oral contraceptives were reported to have higher prolactin levels by 12% to 30% (240,241). Some, but not all, studies suggest that there is a dose-dependent effect (4). No increase in basal prolactin levels is reported during therapy with modern contraceptives with lower amounts of estrogen (242)or estrogen plus cyproterone acetate alone (243).

In transgender patients, estradiol or ethinyl estradiol treatment, the prolactin level rise was dependent on the dose of estrogen, duration of exposure, and alteration of SHBG levels. Estradiol infusion at levels above 10,000 pg/mL for as short as 6-7 hours significantly elevated prolactin levels by 3- to 4-fold, whereas ethinyl estradiol 2 mg/day for 1 month did not consistently elevate prolactin in all patients, which can be due to its ability to increase SHBG binding and maintaining free portion in the normal range (114) (Table 1, 3).

For women on post-menopausal hormone replacement therapies over 2.5 years, serum prolactin measured were within the normal range (244). In another study on 75 women, who were randomly assigned to three groups: control (receiving placebo), transdermal hormonal replacement (biphasic 17β-estradiol and progesterone, natural hormones), and oral ethinyl-estradiol and desogestrel, prolactin levels significantly increased in the oral group, but not in the transdermal group. There was a significant difference in hormone levels: in the oral group, estradiol levels increased five times and estrone levels eleven times. In the transdermal group, estrone and estradiol levels were increased three times (245).

GONADOTROPHINS AND GNRH AGONISTS

In addition to the known prolactin function in lactation, several studies have suggested other benefits of prolactin in oocyte development, formation of corpus luteum and its survival, steroidogenesis and implantation (246). In natural cycles there is a transient increase in late follicular phase of prolactin, but this increment is higher in stimulated cycles (246). In a cohort study were included 79 patients; 60 individuals underwent in vitro fertilization, 14 received clomiphene citrate treatment, and five patients with premature ovarian failure were administered estradiol. During the course of human menopausal gonadotrophin (hMG) treatment, a notable increase in both serum estradiol and prolactin concentrations were observed from early to late follicular days (P < 0.01). Specifically, prolactin levels increased from an initial mean value of 367±38 mIU/L (17.25±1.8 ng/mL) to 991±84 mIU/L (46.6±4 ng/mL) (Table 1). Bromocriptine effectively mitigated the increase in prolactin levels but was associated with a significant elevation in estradiol levels (P < 0.05) because prolactin itself works as a controller of estradiol increment. Clomiphene treatment led to a significant increase in serum estradiol levels (P < 0.01) but a significant decrease in serum prolactin concentrations during the late follicular phase (P < 0.01), indicating disruption of the estradiol-prolactin feedback mechanism. Among patients with premature ovarian insufficiency, serum prolactin concentrations increased concomitantly with rising serum of estradiol concentrations (after estradiol administration). Additionally, it was observed that the presence of prolactin significantly reduced estradiol production by granulosa cells (P < 0.05) (116).

An increment of prolactin levels is found even after hCG administration with a maximum prolactin level of 93.2 ng/mL; 1983 mIU/L (115).Notably, knowing that prolactin is a stress hormone, during assisted procedures it is increased, but this is a transitory increment without consequences in fertility outcome (247).

Not only gonadotrophins but also GnRH agonists are widely used during invitro fertilization to maintain a controlled and synchronized ovarian stimulation. Use of leuprolide acetate (GnRH agonist) concomitantly with hMG, resulted in higher prolactin and estradiol levels in comparison with patients receiving only hMG (prolactin 24.2 vs 16.8 ng/mL; 515 vs 358 mIU/L) (117). In another randomized study, along protocol with 0.1 mg subcutaneous triptorelin starting from day 10 of the preceding stimulation cycle and short protocol, where 0.1 mg subcutaneous triptorelin is given in the stimulating cycle, were compared. Prolactin levels were measured at 9 am in the first day of hCG administration. The long protocol correlated with higher prolactin levels (31.3 ± 16.9 vs 23.7 ± 11 ng/mL; 666 ± 359 vs 504 ± 234 mIU/L) (248).

In children, GnRH agonists are used in precocious puberty (CPP)as well as growth hormone deficiency (GHD)who do not properly respond to exogenous growth hormone treatment. In a study involving 119 children with CPP and 93 with GHD, treated with triptorelin or leuprolide, prolactin levels were measured before and every six months for 6 years for CPP group and for 2 years for GHD group. Moreover, prolactin levels were checked after 6 and 12 months of treatment withdrawal. In this study was concluded that even though prolactin levels were higher in triptorelin treated patients (only 3.8% developed hyperprolactinemia in triptorelin group which was solved after withdrawal – baseline 12.5 ± 3.7 ng/mL (266 ± 79 mIU/L) to max 45.6 ± 4.5 ng/mL; 970 ± 96 mIL/L), no significant difference was found in prolactin in basal condition and during GNRH agonist treatment in CPP and GHD (190)(Table 2).

OTHER DRUGS

A lot of other drugs have been reported to cause mild (less than 2-fold increment) increases in prolactin levels. A synthesized visualization of these mechanisms is shown in Figure 2.

The acute administration of buspirone, an anxiolytic medication, was investigated in a study involving 8 healthy volunteers. The findings revealed an increase in plasma prolactin levels across all participants compared to the baseline levels observed in 8 control subjects. During the study, blood samples were collected at 30-minute intervals over a duration of 2 hours. The zenith of prolactin levels was observed between minutes 90 and 120 for all individuals, with the maximum elevation reaching 37 ng/mL (787 mIU/L) (249). It is noteworthy that the augmentation of prolactin is believed to exhibit a dose-dependent relationship. Furthermore, it was observed that chronic usage of buspirone did not lead to significant alterations in prolactin levels, indicating a potential adaptation to the acute changes induced by the medication. The underlying mechanism responsible for this phenomenon is posited to involve both serotoninergic and dopaminergic implications (119).

Carbamazepine, a widely used anticonvulsant, was examined in a cohort comprising 4 patients with complex partial seizures undergoing chronic carbamazepine treatment (200 mg administered three times daily). Blood samples were collected at intervals of 2 hours. Additionally, a group of 5 patients with untreated epileptic seizures participated, wherein a thyrotrophin-releasing hormone (TRH) stimulation test was performed both prior to and 35-50 days post the administration of 200 mg carbamazepine three times daily. Blood samples were obtained 10, 30, and 60 minutes following intravenous injection of 200µg TRH. Furthermore, 4 normal volunteer subjects were included in the study. On the first day, a placebo was administered, followed by the administration of 400 mg carbamazepine at 8 AM on the second day. Blood samples were collected at baseline on both days and subsequently at hourly intervals until 4 PM. After a span of two weeks, a nocturnal study was conducted, spanning from 6 PM to 6 AM. The investigation revealed that there were no discernible alterations in spontaneous prolactin release or TRH-stimulated prolactin levels. However, a slight increase in sleep-entrained prolactin values was observed, while retaining the secretory circadian rhythm. Given that the release of prolactin during sleep is largely attributed to serotoninergic activity, it is plausible that the modest increment (less than 2-fold) may implicate serotoninergic modulation (working as a serotonin-releasing factor and reuptake inhibitor) facilitated by carbamazepine (120,121).

Sympathomimetic amines fenfluramine and sibutramine, formerly used for appetite suppression due to their stimulatory effect on the synaptic concentration of serotonin, have been shown to induce hyperprolactinemia as a result of increased serotoninergic activity and postsynaptic stimulation of 5HT2Areceptors. In a case report, after starting sibutramine, a 38-year-old female patient developed hyperprolactinemia (prolactin levels 46 and 89.6 ng/mL (978 and 1906 mUI/L) with amenorrhea and galactorrhea. Discontinuation of sibutramine, confirmed by a sella MRI, led to rapid normalization of prolactin levels within 15 days, and symptoms resolved during a 90-day follow-up (2,127).

Cholinomimetic drugs have been reported controversially in the literature regarding their ability to cause hyperprolactinemia. However, in collaborative studies from the National Institute of Mental Health and the University of California, San Diego, three separate experiments were conducted involving volunteers of different genders and ages. In the first experiment, nine volunteers received physostigmine salicylate at 33 µg/kg, while in the second experiment, eleven male volunteers were given 22 µg/kg of physostigmine salicylate. The third experiment involved six volunteers receiving 3 mg of arecoline hydrobromide. Placebo saline was administered in all experiments as well. It was shown that intravenous injection of physostigmine or arecoline can elevate prolactin correlating with raised β-endorphin levels in the blood. Prolactin elevation was less than 100 ng/mL (2127 mUI/L). Cholinergic activation in the hypothalamus, particularly focusing on β-endorphin, might help in explaining how peptides modify primary neurochemical effects on hormone regulation in the hypothalamus and pituitary (44).

Bucillamine, an analogue of D-penicillamine used as an antirheumatic drug in Japan, has been reported to induce hyperprolactinemia (109 ng/mL (2319 mUI/L)) after 30 months of treatment start, associated with gynecomastia and galactorrhea in one case report. The mechanism remains unclear (133).

‘Ecstasy’ (MDMA) was shown to increase prolactin secretion in rhesus monkeys by stimulating serotonin release and by direct-acting as a 5HT2A agonist (250); In nine studies, five of them observed an increase in prolactin levels due to the intervention. However, in the remaining studies, there was no significant change in prolactin levels, and these unresponsive results tended to occur when a lower dose of the intervention was used on average. This suggests a potential relationship between the dosage of the intervention and its effect on prolactin levels (135).

Smoking, particularly the consumption of high-nicotine cigarettes, has been associated with a significant acute elevation in prolactin levels, ranging from 50% to 78% above the baseline, within 6 minutes after smoking. These elevated levels persist for approximately 42 minutes and return to baseline within 120 minutes of initiating smoking (136). The underlying mechanism probably involves the stimulation of rapid prolactin release through the augmentation of endogenous opioids, which subsequently inhibits dopamine release (251). However, prolonged nicotine exposure leads to desensitization of dopamine receptors, and lowers dopamine turnover (48) probably contributing to hyperprolactinemia. It has been hypothesized that the increased incidence of osteopenia and osteoporosis could be at least partly related to this effect (252).

Recently, an association has been reported between HIV-1 protease inhibitors and the adverse effect of galactorrhea and hyperprolactinemia in four HIV-1 infected women treated with indinavir, nelfinavir, ritonavir, or saquinavir. The cause of this unexpected toxicity could be attributed to several possible mechanisms: 1) Protease inhibitors may enhance the stimulatory effects of prolactin due to their inhibition of the cytochrome P450 system, leading to longer half-life of prolactin; 2) opportunistic infections in AIDS patients may induce cytokine-driven prolactin production by pituitary or immune cells; 3) protease inhibitors might exert direct endocrine effects on the pituitary or hypothalamus (253,254). To explore mechanisms of hyperprolactinemia induced by protease inhibitors, experiments were conducted using rat pituitary cells and hypothalamic neuronal endings. The results showed that both ritonavir and saquinavir could directly stimulate prolactin secretion, while not affecting dopamine release. This suggests that these protease inhibitors might interact with specific mammalian proteins in the anterior pituitary involved in prolactin secretion, leading to the observed galactorrhea and hyperprolactinemic effect (137).

Regarding chemotherapy and immunosuppression, there are some controversial data on the effect of chemotherapy and immunosuppression on prolactin levels, as significant prolactin increases are not frequent and usually mild (2). Prolactin and growth hormone have been involved as part of a cytokine system in the recovery of the immune response after chemotherapy and bone marrow transplantation (255). In a study of 20 breast cancer patients undergoing high-dose chemotherapy and autologous stem-cell transplantation, plasma prolactin levels increased within and 30 days after transplant, yet still remaining within the normal range. The use of antiemetic drugs further raised prolactin levels. Patients in continuous complete remission after transplantation exhibited higher prolactin levels, while elevated prolactin did not impact disease-free survival, suggesting potential for further research into post-transplant immune response (256).

Radiotherapy for intracranial germ cell tumors was shown to induce hyperprolactinemia with a prevalence of 35.3% (138).

Other drug inducing hyperprolactinemia are described in Table 1.

DRUGS REPORTED TO DECREASE PROLACTIN LEVELS OR HAVE AN EQUIVOCAL EFFECT

Several medications, beyond the established treatments like cabergoline, bromocriptine, carbidopa and levodopa, have reported effects on reducing prolactin levels. For instance, pseudoephedrine, an α-adrenergic stimulant primarily affecting α1 receptors, shares structural similarities with amphetamine and moderately stimulating dopamine release in the brain by acting on D2 receptors in the pituitary, consequently lowering prolactin levels. Studies have indicated pseudoephedrine's potential to decrease milk production, at least partly attributed to its effect on prolactin levels through dopaminergic actions in the pituitary (132). Moreover, indirect evidence suggests that α-1 receptors stimulation leads to decreased prolactin levels (41).

Amphetamine was seen to produce a poor prolactin suppressant effect in either normal- or hyperprolactinemic subjects. The proposed mechanism of prolactin lowering potential is due to their ability to stimulate the release of dopamine (257). However, during the withdrawal period of cocaine use, hyperprolactinemia has been observed, probably due to a decrease in dopamine levels, leading to dysregulation in the dopamine system and increased prolactin. Moreover, during withdrawal, prolactin can be secreted as a stress hormone (129).

Guanafascine, an α2 adrenergic agonist, used to treat ADHD, has been shown to decrease prolactin levels. In a longitudinal study spanning three years involving 15 patients diagnosed with hyperprolactinemia, the noteworthy suppressive impact of guanfacine on prolactin levels suggests potential involvement of hypothalamic or extrahypothalamic adrenergic pathways in the intricate regulation of prolactin secretion (131). Even though α2 stimulation has been shown to increase prolactin levels in rats, this is not fully understood in humans making the explanation in this case confusing (42).

The impact of benzodiazepines (BDZ) on prolactin secretion is a subject of debate. Research findings have yielded conflicting results. Some studies conducted on both non-epileptic patients and healthy volunteers have not detected significant modifications in prolactin levels following BDZ treatment (258). A study on 30 adolescent patients with schizophrenia with gradually increasing doses of diazepam to a maximum of 100-400 mg/day, with 4 weeks of treatment, showed that only doses higher than 250 mg/day give a significant but mild increase in prolactin levels. Proposed mechanisms are inhibition of TIDA neurons by activation of the GABA system, or activation of the endorphin-ergic system leading to hyperprolactinemia (118). On the contrary, diazepam was found to suppress the secretion of prolactin in vitro through one of two mechanisms: it either strengthens the direct inhibitory action of GABA on prolactin release, or it hinders a benzodiazepine-sensitive Ca2+-calmodulin dependent protein kinase at micromolar concentrations leading to a reduction of prolactin secretion (259).

Moreover, phenytoin, an anticonvulsant impeding sodium channels in nerve cells, have generated conflicting data regarding their impact on prolactin levels. In animal studies, phenytoin showcased a rapid decline in both prolactin release and mRNA concentrations, functioning as a partial T3 agonist by binding to T3 nuclear receptors (260). However, clinical observations revealed elevated resting levels of prolactin in phenytoin-treated patients compared to untreated counterparts. Remarkably, responses to metoclopramide and bromocriptine remained unaltered, indicating a limited effect of phenytoin on the D2 receptors present on lactotrophs (261). Even the conclusions drawn from these findings remain contentious. Evidence suggests that phenytoin treatment may enhance the growth hormone response to levodopa, implying a phenytoin-induced dopaminergic activity at the hypothalamic-pituitary level (122). More specifically, it is postulated that phenytoin might enhance dopamine receptor sensitivity by inhibiting the Ca2+ calmodulin complex. This effect could contribute to reduced prolactin secretion (122). On the contrary, other studies have not demonstrated any notable alterations in prolactin levels due to phenytoin administration (262). The discordant outcomes surrounding phenytoin's impact on prolactin levels underscore the complexity of its effects and necessitate further investigation for conclusive insights into its mechanisms of action.

In another comprehensive study involving 126 subjects, both males and females, with generalized or partial epilepsy receiving phenobarbital as monotherapy or in combination with phenytoin or benzodiazepines, a distinct pattern emerged. Specifically, the administration of phenobarbital, either alone or in combination, resulted in elevated prolactin levels, but this elevation was found to be statistically significant only in the male participants. Notably, knowing that an epileptic attack itself can cause hyperprolactinemia, those data remain confusing. The proposed mechanism in this study is phenobarbital interaction with GABA receptors, leading to increased prolactin levels (263). However other studies do not show any change in prolactin levels (123).

Valproic acid, an anticonvulsant working as a central stimulant of GABAergic neurons, has demonstrated the ability to reduce prolactin basal levels as well as TRH-stimulated prolactin levels. This is indirect proof of the synergically acting of GABA neurons with dopaminergic tracts (124). However, in another study, no effect of valproic acid on prolactin levels was noticed during the night (264).

Lithium carbonate, a pharmaceutical agent employed as a mood stabilizer, has undergone investigation in different studies, as it decreases dopamine release and glutamate, and increases inhibitory GABA (265). One of them encompassed a longitudinal examination involving 9 patients diagnosed with bipolar disorder. The focus of this study was the assessment of plasma prolactin levels before and 12 hours after the evening administration of lithium. Evaluations were conducted on days 1, 6, 8, 13, 30, 60, and 90. Notably, this investigation yielded no discernible correlation between lithium concentration and prolactin levels, and no statistically significant alterations in prolactin levels were observed. The second part of this study adopted a cross-sectional design, involving 26 patients with an established history of long-term lithium treatment spanning durations of 3 months to 20 years. A comparative analysis revealed that prolactin levels, measured at 9 AM following a one-hour period of rest, did not demonstrate elevation in comparison to 16 controls. It is noteworthy that in both studies, lithium concentrations ranged from 0.4 to 1.4 mmol/L (normal range 0.5-1.2 mmol/L) (266). Additionally, the administration of lithium did not exert an impact on the plasma prolactin response to thyrotrophin-releasing hormone (TRH) stimulation compared to pre-treatment levels (267). The combined findings from these investigations provide compelling evidence that lithium does not contribute to hyperprolactinemia, thereby distinguishing it from medications with such an effect.

Cocaine has been shown to decrease prolactin levels beginning at 30-min following cocaine administration reaching statistical significance at the 90- and 120-minute time points (134).

Those medications are mentioned in Table 1. Their mechanism of altering prolactin levels is summarized in Figure 2.

HERBAL MEDICINES AFFECTING PROLACTIN LEVELS

In Table 4 is list of herbal medicines has been used traditionally to stimulate lactation (268). However, firm scientific evidence that they actually induce hyperprolactinemia is scarce.

Table 4. Lactogenic Herbs (268)
Family name	Species name	Common name
Amaryllidaceae	Allium sativum	Garlic
Annonaceae	Xylopia aethiopica	African Pepper or Ethiopian Pepper
Asclepiadaceae	Secamoneafzelii	-
Costaceae	Costusafer	African Ginger
Euphorbiaceae	Euphorbia hirta	Asthma Plant or Tawa-Tawa
	Euphorbia thymifolia	Petty Spurge
	Hymenocardiaacida	African Almond or Honeytree
	Plagiostylesafricana	-
	Ricinus communis	Castor Bean Plant
Leguminosae	Tamarindus indica	Tamarind
	Acacia nicolita	-
	Desmodiumadscendens	-
Malvaceae	Hibiscus sabdariffa	Roselle or Red Sorrel
Malvaceae	Gossypium herbaceum	Cotton Plant
Moraceae	Milicia excelsa	African Teak or Iroko
Moraceae	Ficus species	Ficus or Fig trees
Musaceae	Musa paradisiaca	Plantain
Ranunculaceae	Nigella sativa	Black Cumin or Black Seed
	Actaea (Cimiciguga) racemose	Black Cohosh
Solanaceae	Solanum torvum	Turkey Berry or Devil's Fig
Verbanaceae	Lippia multiflora	Bush Tea or False Green Tea
Zingiberaceae	Aframomummelegueta	Grains of Paradise or Alligator Pepper
Fabaceae	Trifolium pratense	Red Clover
Fabaceae	Trigonella foenum-graecum	Fenugreek
Apiaceae	Foeniculum vulgare	Fennel

Some herbs are known to decrease prolactin levels. For example, chaste tree (Vitex agnus-castus) decrease prolactin levels by activating to D2-receptors and suppressing prolactin release, as shown in in vitro experiments on lactotroph cell cultures and in in vivo animal experiments (269). Another herb, Mucuna pruriens, which is a natural source of l-dihydroxyphenylalanine (a dopamine precursor) is found to decrease prolactin levels in humans (270). Vitamin B6 (pyridoxine), by acting as a coenzyme in dopamine synthesis and aspartame, a sweetener metabolized in phenylalanine (dopamine precursor), have been shown to interfere with milk production by reducing prolactin levels (271). Ashgawanda (Withania somnifera) is found to decrease prolactin levels up to 12% (272). Moreover, oral zinc is found to decrease prolactin levels below the normal range in all 17 subjects with normal prolactin levels, in scenario of increased zinc levels in the blood (273).

While none of the mentioned herbs are currently established within clinical guidelines for specifically lactogenic or prolactin-reducing purposes, ongoing research and anecdotal evidence suggest potential roles for these botanicals as adjunctive therapies.

CONCLUSION

In summary, this review underscores the significant role of drug-induced hyperprolactinemia in causing higher prolactin levels and provides detailed insights into how pharmaceutical agents contribute to this effect. However, understanding the complex mechanisms behind drug-induced hyperprolactinemia is still a work in progress. More research is needed to delve deeper into these mechanisms and gain better insights. These efforts will contribute to refining treatment strategies and improving patient care.

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Familial Hypercholesterolemia

ABSTRACT

Familial hypercholesterolemia (FH) is a prevalent, autosomal co-dominant disorder of lipid metabolism that results in elevated low-density lipoprotein cholesterol (LDL-C) levels and premature atherosclerosis. Screening for and identifying heterozygous FH in childhood is critical, given its high prevalence and asymptomatic presentation. Furthermore, treatment of FH in childhood is effective at lowering LDL-C levels and has the potential to reduce atherosclerotic cardiovascular disease (ASCVD) events in adulthood. Selective screening based on family history had previously been recommended to identify children and adolescents with FH or other lipid disorders. However, studies indicated that many individuals with heterozygous FH were missed with this approach, and therefore in 2011 the National Heart, Lung, and Blood Institute Expert Panel recommended universal screening of children and adolescents between ages 9 and 11 years and again at ages 17 to 21 years, in addition to selective screening, in order to identify pediatric individuals with heterozygous FH. This approach was affirmed in the 2018 American College of Cardiology and the American Heart Association (ACC/AHA) Cholesterol Guidelines, along with endorsing cascade screening as another reasonable approach to identifying children with FH. Once FH is diagnosed, prompt treatment with lifestyle modification should be initiated. When lifestyle interventions are not sufficient, pharmacotherapy using statins has been shown to be effective at lowering LDL-C, generally safe in short and medium- term studies, and may be beneficial at reducing ASCVD events. Other medications can be useful at lowering LDL-C in conjunction with statin therapy, although generally statins are sufficient in young patients. Homozygous FH is a rare disorder manifesting as extremely high LDL-C and ASCVD in childhood, requiring aggressive multimodal management. Overall, studies are needed to determine the optimal timing and intensity of statin therapy, and to better understand long-term safety and ASCVD outcomes in adulthood for lipid-lowering pharmacotherapy initiated in pediatric patients with heterozygous FH.

INTRODUCTION

Familial hypercholesterolemia (FH), as classically described, is the most common single gene disorder of lipoprotein metabolism and causes severely elevated low-density lipoprotein cholesterol (LDL-C) levels. The prevalence of FH is 1 in 200 to 1 in 300 individuals of different ethnicities (1,2), and it is strongly associated with premature coronary artery disease (CAD) (3). Data from observational studies suggest that untreated FH is associated with ~90-fold increase in mortality due to atherosclerotic cardiovascular disease (ASCVD) in young adults (4). Since early treatment may significantly reduce CAD-related morbidity and mortality in individuals with heterozygous FH (5), early identification and intervention during childhood may greatly improve outcomes in adulthood.

EPIDEMIOLOGY

The prevalence of FH varies substantially, depending upon the criteria used to define the disorder and the ancestry of the population. Previously, the prevalence had been described as 1 in 500 individuals based on early work by Drs. Brown and Goldstein (6,7). More recent analysis of white European populations, which tend to be less ethnically and racially diverse than the US, show higher prevalence rates (8,9). An analysis using a modified version of the Dutch Lipid Clinic (DLC) criteria applied to participants in the 1999 to 2012 National Health and Education National Surveys (NHANES), a nationally representative survey of the US population, suggest FH affects 1 in 250 US adults (1). The prevalence of FH in US children and adolescents is not as well characterized, although presumably it is similar. Some estimate that as few as 10% of individuals with FH have been identified in the US.

PATHOPHYSIOLOGY AND GENETICS

FH was initially defined by Brown and Goldstein as a disorder or defect in the LDL receptor (LDL-R) (3). More recently, the description of FH has been expanded and used to describe any defects in LDL-C processing and/or signaling that may lead to a phenotype characteristic of FH (10). FH may be more common and complicated than previously thought, with many different genetic variants leading to pathogenesis. Overall, nine genes are causative for autosomal forms of FH, and up to 50 polymorphic loci contribute to polygenic susceptibility to elevated LDL- cholesterol levels (11). Some etiologies for the FH phenotype include defects in apoB100 lipoprotein, the major atherogenic lipoprotein component of LDL-C, as well as gain of function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9), which promotes the degradation of the LDLR, resulting in reduced LDL-C clearance (7). Gene mutations in LDL-R, the apolipoprotein B gene, or in PCSK9 occur in approximately 93, 5, and 2 percent of individuals with a phenotype consistent with FH, respectively (12). The associated impairment in function of these receptors or proteins results in overall reduced clearance of LDL particles from the circulation and elevation in plasma LDL-C. There is also increased uptake of modified LDL by macrophage scavenger receptors, resulting in lipid accumulation in macrophages and foam cell formation, a precursor to atherosclerotic plaque development (13). Thus, the typical lipid profile of FH is characterized by elevated LDL-C (as high as 300 mg/dL) with subsequently increased total cholesterol (TC) levels; in general, triglycerides are normal, and high-density lipoprotein (HDL-C) can be low or normal. FH has an autosomal dominant inheritance pattern which results in hypercholesterolemia and early ASCVD events.

There are various genetic mutations that can cause FH, which can either be monogenic or polygenic in nature. See Table 1 below for a list of genes that are associated with FH.

Table 1. Genes Associated with FH
Monogenic
Autosomal Dominant LDLR APOB PCSK9 APOE*
Autosomal Recessive LDLRAP1 LIPA (Lysosomal acid lipase deficiency) ABCG5 and ABCG8 (Sitosterolemia)
Polygenic
Genetic variants associated with FH which together can impact LDL-C levels

*Specific mutations or candidate regions associated with FH

**Refer to specific Endotext sections on lysosomal acid lipase deficiency and sitosterolemia for more information

In general, homozygotes for mutations in the LDL-R gene are more adversely affected than heterozygotes, reflecting a “gene dosing effect” of inheritance. Unless there is consanguinity in a family in which heterozygous FH is present, homozygous FH is less prevalent and may affect as many as 1 in 160,000–300,000 individuals (14). Additionally, the homozygous FH phenotype can be seen in compound heterozygotes which can occur in offspring of unrelated parents due to a different disease-causing mutation on each allele. The severity of the FH phenotype does not necessarily depend upon the presence of true homozygosity or compound heterozygosity inheritance; rather it is determined by the degree of disturbance in LDL metabolism. For additional information on the genetics as well as pathophysiology, refer to “Familial Hypercholesterolemia: Genes and Beyond” by Warden et al., www.endotext.org (15).

FH PHENOTYPE

Clinical Symptoms

HOMOZYGOUS FH

Due to the excessively high plasma LDL-C levels in homozygous FH, cholesterol deposits are common in the tendons (xanthomas) and eyelids (xanthelasmas), and generally appear by one year of age. Tendon xanthomata are most common in the Achilles tendons and dorsum of the hands but can occur at other sites. Tuberous xanthomata typically occur over extensor surfaces such as the knee and elbow. Planar xanthomas may occur on the palms of the hands and soles of the feet and are often painful. Xanthelasmas are cholesterol-filled, soft, yellow plaques that usually appear on the medial aspects of the eyelids. Corneal arcus is a white or grey ring around the cornea (16).

HETEROZYGOUS FH

In addition to increased serum cholesterol and risk for premature coronary artery disease (see below), patients with heterozygous FH may have tendon xanthomas and corneal arcus that appear after the age of 20 years (16).

LDL-C Levels

In clinical practice, there is not a universal LDL-C threshold that determines a diagnosis of FH. Generally, the level of LDL-C that warrants further evaluation depends upon the age of the patient and whether additional family members have known hypercholesterolemia and/or early ASCVD. As suggested by recent guidelines (7,17), children and adolescents with a negative or unknown family history and LDL-C level persistently ≥190 mg/dL (4.9 mmol/L) suggests FH; in patients with a positive family history of hypercholesterolemia or early ASCVD, an LDL-C level of ≥160 mg/dL(4.1 mmol/L) is consistent with FH (see Table 2).

Table 2. Acceptable, Borderline, and High Lipid Levels for Children and Adolescents
Lipid	Low (mg/dl)	Acceptable (mg/dl	Borderline-High (mg/dl)	High (mg/dl)
TC		<170	170-199	>200
LDL-C		<110	110-129	>130
Non-HDL-C		<120	120-144	>145
Triglycerides 0-9 years 10-19 years		<75 <90	75-99 90-129	>100 >130
HDL-C	< 40	>45

Adapted from the NCEP expert panel on cholesterol levels in children (14)

Cardiovascular Disease

Cardiovascular risk in FH patients is determined by both the LDL-C concentration and by other traditional risk factors. Homozygotes have early-onset atherosclerosis, including myocardial infarction, in the first decade of life (reported as early as age two years), and are at increased risk for CAD-related mortality in the first and second decades (16). Additionally, patients with homozygous FH can develop cholesterol and calcium deposits that can lead to aortic stenosis and occasionally to mitral regurgitation.

Heterozygotes are also at increased risk for early-onset CAD between the ages of 30-60 years (18). Children with heterozygous FH have thicker carotid intima-media thickness (cIMT), an anatomic measure of arterial thickness associated with atherosclerosis, compared to unaffected siblings and healthy controls (19,20). One study showed those treated with statin medications (HMG-CoA reductase inhibitors) at younger ages had less carotid atherosclerosis compared to the placebo group. Results from long-term studies of statins in children with FH are just emerging, and indicate that statin treatment during childhood may slow progression of cIMT and reduce the risk of CVD in adulthood (21).

SCREENING

Given the high prevalence of FH and the improved outcomes with early treatment, pediatric lipid screening has become very important for the detection of FH. However, the approach to lipid screening in childhood and adolescences has varied over the past decades and is somewhat controversial, with the US Preventative Services Task Force (USPSTF) recently concluding that that the current evidence is insufficient to assess the balance of benefits and harms of screening for lipid disorders in children and adolescents (22). Selective screening of young individuals with a family history of hypercholesterolemia and/or early CV events or patients at risk for atherosclerosis for other medical conditions has been recommended for several decades (23–25). However, screening individuals based only on family history may miss 30-50% of children with elevated LDL (24–26). Thus, the 2011 Expert Panel, supported by the more recent 2018 ACC/AHA Cholesterol Guidelines (27), recommended universal lipid screening, which involves screening in childhood at two time points, once between ages 9 and 11 years, and then again between ages 17 and 21 years (28). Universal screening is recommended in those not already selectively screened based on family history or personal risk factors (Table 3).

Selective screening for FH involves obtaining a fasting or non-fasting lipid panel in individuals ages 2 to 21 years with:

Family history of early atherosclerosis or high cholesterol.
Relatives of individuals with identified FH.

Lipid testing should also be performed in the presence of risk factors or medical diagnoses that increase risk for CVD (including hypertension, current cigarette smoking, body mass index ≥ 85^th percentile, diabetes mellitus type I and II, chronic kidney disease/end-stage renal disease, chronic inflammatory diseases, human immunodeficiency virus infection, and nephrotic syndrome) (28).

Universal screening involves obtaining either a fasting lipid profile or a non-fasting non-HDL, (calculated by subtracting HDL from TC) in childhood at two time points:

Between ages 9-11 years.
Between ages 17-21 years.

Table 3. Screening for Hypercholesterolemia
Approach	Age in Years	Population
Selective	2-21	Family history of early atherosclerosis or high cholesterol Presence of risk factors or medical conditions that increased early CVD risk*
Universal	9-11 and 17-21	All

Table 3. Screening for Hypercholesterolemia

Approach

Age in Years

Population

Selective

2-21

Family history of early atherosclerosis or high cholesterol

Presence of risk factors or medical conditions that increased early CVD risk*

Universal

9-11 and 17-21

All

*Selective screening is indicated in individuals with hypertension, current cigarette smoking, body mass index ≥ 85^thpercentile, diabetes mellitus type I and II, chronic kidney disease/end-stage renal disease, chronic inflammatory diseases, human immunodeficiency virus infection, and nephrotic syndrome

DIAGNOSIS

The diagnosis of FH can be made clinically and through genetic testing; genotype needs to be interpreted in the context of phenotype. For heterozygous FH, the clinical diagnosis is made based on the presence of high levels of total and LDL cholesterol in combination with one or more of following (17):

Family history of hypercholesterolemia (especially in children) or known FH.
History of premature CAD in the patient or in family members.
Physical examination findings of abnormal deposition of cholesterol in extravascular tissues (e.g., tendon xanthoma), although these rarely occur in childhood.

There are several clinical scoring systems used to diagnose FH, and these vary based on the weight given for each diagnostic criteria (11). In general, clinical diagnosis of homozygous FH can be made in individuals with the following criteria (14):

Untreated LDL-C >500mg/dL (>13 mmol/L) or treated LDL-C ≥300 mg/dL (>8 mmol/L), AND
Cutaneous or tendon xanthoma before age 10 years, OR
Elevated LDL-C levels consistent with heterozygous FH in both parents.

There are various disease states or other factors that can increase LDL-C levels and should be considered when diagnosing FH. Some of these include the following:

Obesity
Hypothyroidism
Diabetes mellitus
Nephrotic syndrome
Chronic renal failure
Cholestasis
Biliary atresia
Hepatitis
Biliary cirrhosis
HIV infection/AIDS
Various drugs/medications
Alcohol
Pregnancy
Very low carbohydrate ketogenic diets

Genetic Testing

Identifiable gene defects in LDLR, APOB, or PCSK9 have been identified in 60 to 80% of individuals with a heterozygote FH phenotype. Genetic testing has not routinely been performed in the clinical setting due to concerns about cost to the patient and because it was not likely to alter management, given that treatment decisions were usually based on LDL-C levels. However, FH genetic testing has recently been recommended to become the standard of care for patients with definite or probable FH, and the rationale for such testing includes the following: 1) facilitation of definitive diagnosis of FH lowers the concern for other secondary causes of high LDL-C; 2) pathogenic variants correlate with higher cardiovascular risk, which indicates the potential need for more aggressive lipid lowering; 3) increase in initiation of and adherence to therapy; and 4) cascade testing of at-risk relatives (29). Overall, the clinical significance of normal or moderately elevated LDL-C levels in the setting of a genetic defect in the LDLR or other possibly pathogenic defects is unknown.

TREATMENT

The guidelines for initiating treatment in patients with the FH phenotype are based on age, severity of LDL-C elevation, as well as family and medical histories. Lifestyle therapy is recommended for all children and adolescents with LDL-C levels ≥ 130 mg/dL. If lifestyle intervention is insufficient, medications can be considered in children beginning at age 10 years, or as early as age 8 in high-risk patients and in the presence of a very high-risk family history. For healthy children and adolescents ages 10-21 years, lifestyle therapy should be provided to those with an LDL-C ≥ 130 mg/dL, and medication should be initiated if LDL-C remains ≥ 190 mg/dL despite 6 or more months of lifestyle modification. If there is a family history of early atherosclerotic disease, then medication should be started in individuals with LDL-C levels ≥ 160 mg/dL who do not respond sufficiently to lifestyle modification. If an individual has a high-risk medical condition, as noted above, medication can be considered for those with a persistently elevated LDL-C ≥ 130 mg/dL. In general, the goal of treatment is to maintain an LDL-C level ≤ 130 mg/dL or ≥ 50% reduction in LDL concentration; lower ranges may be considered in high-risk patients. Medications should be initiated in all patients with homozygous FH at the time of diagnosis, regardless of age, and additional treatments should also be considered.

Lifestyle Treatment

The mainstay of treatment for pediatric lipid disorders is lifestyle modification. A low saturated fat diet, without trans-fat, and high in fruits and vegetables, is the recommended diet for lowering LDL-C. This dietary approach has been shown to be both safe and beneficial in the general pediatric population (28,30,31). Additionally, nutritional and physical activity interventions have been shown to lower LDL-C and improve CVD risk factors in children with obesity in meta-analyses (32). Despite this, in adults with FH, lifestyle modifications have been shown to only lower LDL-C modestly (33). Furthermore, the effect of physical activity on LDL-C levels has not been well studied in children with FH.

Pharmacotherapy

STATINS

The majority of patients with FH are treated with medications, and statins are the recommended first line pharmacotherapy. In a Cochrane meta-analysis of pediatric patients with FH, statins were shown to lower LDL-C by 32% (34). Furthermore, more intensive statin therapy in high doses has been shown to lower LDL-C even more significantly, by up to 50% (35,36). Follow-up data from a statin trial in pediatric Dutch patients suggest efficacy and safety, as well as decreased atherosclerosis compared to the subjects’ parents (37). Most recently, the use of statins in children with FH have shown reduced CVD risk in adulthood for these patients compared to their untreated parents with FH, after a 20 year follow-up (21). Although this study was not a controlled or placebo study, it suggests that long-term statin use in childhood may prevent ASCVD compared to not treating.

Several different formulations of statin therapy are available and approved by the US Food and Drug Administration (FDA) for use in children. Treatment is initiated at a low dose (generally 5-20mg depending on the statin potency), which is given once a day, often at night. If needed, the dose is increased to meet the goals of therapy. Side effects with statins are rare, but include myopathy, new-onset type 2 diabetes mellitus (reported in adult primary prevention statin trials), and hepatic enzyme elevation. In pediatric clinical trials, rates of side effects with statin therapy were low and adherence to statin therapy was generally good (38). Side effects of statins are more likely at higher doses and in patients taking other medications, particularly cyclosporine, azole antifungal agents, and other medications and foods (such as grapefruit) that impact the cytochrome P450 system. Adolescent females should be counseled about the possibility of drug teratogenicity and appropriate contraceptive methods while receiving statin therapy. Additionally, providers should be aware that oral contraceptive pills can increase lipid levels.

The NHLBI guidelines recommend the following baseline laboratory evaluation when initiating statin therapy (28):

Fasting lipid profile.
Serum creatine kinase (CK).
Hepatic enzymes (i.e., serum alanine aminotransferase [ALT] and aspartate aminotransferase [AST]).

Screening for type 2 diabetes is also reasonable prior to starting statins. Fasting lipid profiles are repeated at four weeks after the initiation of statin therapy to titrate dose and are repeated every six months in patients on stable therapy. Liver function tests, CK, and hemoglobin A1C should be obtained if signs of adverse effects arise, and may be obtained at regular intervals, for example after dose changes based on best clinical judgement. Ongoing monitoring of growth, other measures of general and cardiovascular health, and review for the presence of additional ASCVD risk factors, such as smoking exposure, should also occur at each visit.

BILE ACID BINDING RESINS

Although bile acid binding resins or bile acid sequestrants have been shown to lower LDL-C by ~10-20% in pediatric trials (39,40), they are often difficult to tolerate given their unpalatability and associated adverse effects (such as bloating and constipation) (41). For these reasons, bile acid binding resins are used relatively infrequently. However, they may be useful in combination with a statin for patients who fail to meet target LDL-C levels (42). The sequestrants are not absorbed systemically, remain in the intestines, and are excreted along with bile containing cholesterol. Therefore, they are considered to be very safe. They can be used in patients who prefer to avoid statins, although they may not lower LDL-C sufficiently to achieve goal levels.

CHOLESTEROL ABSORPTION INHIBITORS (EZETIMIBE)

Ezetimibe is a lipid-lowering mediation that inhibits absorption of cholesterol and plant sterols in the intestines. This agent can be useful in pediatric patients with FH who are not able to reach LDL-C treatment goals on high-intensity statin therapy. Ezetimibe further lowers serum LDL-C and in adults has been shown to improve cardiovascular outcomes without altering the side effect profile (43–45). Specifically in the pediatric population, ezetimibe has been shown to be safe and effective at lowering LDL-C by up to almost 30%, even when used as monotherapy (44,46,47)

PCSK9 INHIBITORS

PCSK9 inhibitors (evolocumab and alirocumab) are human monoclonal antibodies that bind to PCSK9 and promote plasma LDL cholesterol clearance. In Europe, evolocumab is approved in adolescents (≥12 years old) with homozygous FH. In the US, alirocumab is approved only for use in adult patients, and evolocumab is approved for use in adults with heterozygous FH and in homozygous FH, ages 13 and older, who have not responded to other LDL-C lowering therapies. Overall, PCSK9 inhibitors appear to have a good safety and side effect profile in adults (48). These medications have been shown to be very effective, reducing LDL-C by more than 15% in patients with homozygous FH and by 35% in patients with heterozygous FH (49–51). The main disadvantage of PCSK9 inhibitors is that they require injection for administration; cost is also a concern.

Inclisiran is another medical therapy that targets PCSK9 synthesis through a different mechanism. It is a small interfering RNA molecule that triggers the breakdown of messenger RNA coding for the PCSK9 protein. This medication has been recently approved for clinical use and has been shown to be safe and effective in adult patients with heterozygous FH (49). Clinical trials of inclisiran are currently underway in adolescent patients with heterozygous and homozygous FH (50).

EMERGING MEDICAL THERAPIES

There are several promising therapies that aim to reduce LDL-C through different approaches. These include Bempedoic Acid, Lomitapide, and Evinacumab. Bempedoic acid blocks the cholesterol biosynthetic pathway upstream of HMG-CoA reductase through inhibition of adenosine triphosphate citrate lyase. This therapeutic agent has been shown to be effective in treating statin-resistant hypercholesterolemia and in reducing ASCVD in adults (51,52). Bempedoic acid is currently being studied in children with heterozygous FH.

Microsomal triglyceride transfer protein (MTP) plays an essential role in the formation of apoB-containing lipoproteins and has been shown to be inhibited by Lomitapide (53). This drug can reduce LDL-C by approximately 58% and has been approved for use in adults with homozygous FH. So far, Lomitapide has been observed to be safe and effective in pediatric patients with homozygous FH (54).

Evinacumab is a human monoclonal antibody that targets angiopoietin-like 3 (ANGPTL3), which results in the reduction of LDL-C levels via an LDL-receptor independent mechanism (55–57). This drug has been shown to significantly reduce LDL-C in patients with homozygous FH who show little to no LDL-receptor activity and who have had poor response to other treatments (58). Additionally, patients with heterozygous FH have also shown improvements in LDL-C by 50% reduction (59). Evinacumab was approved in early 2021 by the FDA for the treatment of homozygous FH patients starting at age 12 years (60).

Lipoprotein Apheresis

Although statin therapy has been shown to be effective in reducing LDL and prolonging life expectancy in patients with homozygous FH (61), medical treatment alone may not be adequate to achieve recommended treatment goals. Therefore, it is suggested that lipoprotein apheresis (LA) be initiated in patients with homozygous FH as young as 2 years. The efficacy is dependent upon the type of apheresis but can reduce LDL-C by as much as 45 to 80%. Studies in pediatric patients are limited, but there is some evidence suggesting that LA therapy is safe and effective in children with homozygous FH (62).

SUMMARY

FH is an autosomal dominant disorder of LDL metabolism that affects 1 in 200 to 300 individuals. Screening involving lipid measurements, family and medical history, and physical examination is needed to identify affected individuals; cascade screening can be helpful. Lifestyle modification is the first-line therapy for hyperlipidemia in pediatric patients but is usually not sufficient to achieve goal LDL levels. Available evidence suggests that treatment with lipid-lowering pharmacotherapy, such as statins, is effective and generally safe in the short and medium-term. However, further studies are needed to determine the long-term safety and efficacy in preventing ASCVD of lipid lowering medication in pediatric patients with FH.

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Use of Lipid Lowering Medications in Youth

ABSTRACT

The first comprehensive pediatric dyslipidemia guidelines were published by the National Cholesterol Education Program’s Expert Panel on Blood Cholesterol Levels in Children and Adolescents in 1992 and were updated by the American Academy of Pediatrics (AAP) in 1998. In 2008 the AAP issued an updated clinical report detailing recommendations for screening and evaluation of cholesterol levels in children and adolescents as well as prevention and treatment strategies. Since the publication of the first guidelines, rates of pediatric obesity have significantly increased, resulting in a concomitant increase in dyslipidemia. Recently, options for pharmacotherapeutic interventions in pediatric patients have expanded with new FDA approved indications of several lipid lowering medications, as well as additional safety and efficacy data. In 2011, The National Heart Lung and Blood Institute (NHLBI) published its comprehensive report Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents. As with previous guidelines, lifestyle modifications with an emphasis on a heart-healthy diet and daily moderate to vigorous exercise remain an integral part of treatment for pediatric lipid disorders; however, the recommendations for patients requiring management with pharmacotherapy have changed, and will be the focus of this discussion.

INTRODUCTION

The diagnosis, treatment, and monitoring of dyslipidemia in youth has undergone significant transformations in recent years. As detailed by the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) and Bogalusa Heart studies, dyslipidemia plays a vital role in both the initiation, as well as the progression of atherosclerotic lesions in children and adolescents (1-3). Because of their role in premature cardiovascular disease, control of dyslipidemia provides clinicians with an opportunity for reducing morbidity and mortality. Observational data from individuals with genetic mutations that lower atherogenic cholesterol, low-density lipoprotein cholesterol (LDL-C) and non-high-density lipoprotein cholesterol (non-HDL-C), over a lifetime are associated with fewer events and longer life expectancy (4, 5). While these observations are very encouraging, it is not known if achieving the same level of lipid lowering with medications over decades will offer the same protective effects as observed in individuals with life-long lower cholesterol secondary to a genetic mutation (6). Table 1 provides a comparison across the evolution of guideline recommendations for the initiation of pharmacologic intervention with the goal of balancing risk and benefit (7-11). Table 2 details the risk factors and risk conditions described in the NHLBI guidelines (9).

Table 1. Comparison of Recommendations for Treatment
Guidelines	NCEP, AAP – 1992 & 1998			AAP – 2008			NHLBI, AAP – 2011
Pharmacologic Treatment Initiation Parameters*	•	Age > 10 years with LDL-C		•	Age ≥ 8 years with LDL-C		•	Ages 10-21 years with LDL-C
		o	≥ 190 mg/dL		o	≥ 190 mg/dL		o	≥ 190 mg/dL
		o	> 160 mg/dL in addition to a positive family history of premature CVD or presence of at least 2 CVD risk factors in the child/adolescent		o	≥ 160 mg/dL in addition to a positive family history of premature CVD or presence of risk factors		o	160-189 mg/dL in addition to a positive family history of premature CVD or presence of 1 high level risk factor/condition or presence of 2 moderate level risk factors/conditions

					o	≥ 130 mg/dL in addition to presence of diabetes mellitus
						≥ 130 mg/dL in addition to presence of diabetes mellitus
				•	Age < 8 years with LDL-C:			o	130-159 mg/dL in addition to the presence of 2 high level risk factors/conditions or 1 high level and at least 2 moderate level risk factors/conditions
					o	≥ 500 mg/dL		o


							•	Age < 10 years with severe hyperlipidemia or high-risk conditions associated with serious morbidity


*After an adequate trial of diet and lifestyle management.							•	Ages 8-9 years with LDL-C levels consistently ≥ 190 mg/dL in addition to a positive family history OR presence of risk factors



Pharmacologic Medication Recommendations	•	Bile acid sequestrants		•	Bile acid sequestrants		•	Statins
				•	Cholesterol absorption inhibitors
				•	Statins

Table 2. NHLBI Risk Factors and Risk Conditions (9)
NHLBI, AAP 2011 Guidelines: Risk Factors and Risk Conditions
High Level Risk Factors	• Hypertension requiring drug therapy • Tobacco use • BMI ≥ 97^th percentile • High risk conditions
Moderate Level Risk Factors	• Hypertension not requiring drug therapy • BMI ≥ 95^th percentile, < 97^th percentile • HDL < 40 mg/dL • Moderate risk conditions
High Risk Conditions	• T1DM and T2DM • CKD, ESRD, post-renal transplant • Post-orthotopic heart transplant • Kawasaki disease with current aneurysms
Moderate Risk Conditions	• Kawasaki disease with regressed aneurysms • Chronic inflammatory disease • HIV infection • Nephrotic syndrome

PHARMACOTHERAPEUTIC TREATMENT IN YOUTH

The treatment of youth with lipid lowering medications presents some unique challenges and consideration due to their developmental stage, the possibility of extended durations of treatment, and the potential use of concurrent medications that may be counter-productive by increasing lipid levels. As patients progress into adolescence it is particularly important for the patient to understand not only the need for their lipid lowering therapies, but also the consequences of non-compliance. Counseling regarding pharmacotherapy should begin at an early age with developmentally appropriate explanations and expand as patients mature. During adolescence when patients are developing their independence counseling that addresses how to integrate their therapy into their own social norms is important for achieving compliance to both pharmacotherapy as well as lifestyle modifications. Additionally, the cost of therapy significantly impacts compliance and should be factored into therapy decisions especially as youth transition into adulthood and may be faced with changes in insurance coverage. It is also critical to continually readdress the correct use of medications as patients are likely to be on multiple therapies and adolescents will begin some their own medication management as they mature. Regular monitoring for adverse events and side effects of therapy is essential as youth will have a greater lifetime exposure compared to adults and long-term data is generally limited.

As with all pharmacotherapy careful consideration should be given to potential drug interactions including those that may increase lipid levels. It is not uncommon for adolescent patients to be prescribed medications which have the potential to negatively impact lipid levels such as systemic steroids or oral contraceptive pills. Each patient case must be evaluated on an individual basis to determine the risk and benefit of prescribing medications which negatively alter lipid levels for patients also utilizing lipid lowering therapies. It should be noted there is significant risk for adolescent females should they become pregnant while taking lipid lowering medications as some have demonstrated a negative impact on fetal development. Adolescent females should be counselled regarding pregnancy and methods of contraception should be discussed. The US Medical Eligibility Criteria for Contraceptive Use compiled by the CDC details several contraceptive methods where the benefits generally outweigh any theoretical or proven risk for patients with hyperlipidemias (12).

HMG-CoA REDUCTASE INHIBITORS

HMG-CoA reductase inhibitors, or statins, are recommended as first line treatment of youth with severe dyslipidemia who fail non-pharmacologic interventions (i.e., diet and lifestyle modification) (8-11). Statins first debuted in clinical practice in 1987 with the FDA’s approval of lovastatin. At present, there are seven HMG-CoA reductase inhibitors with FDA approval, at varying dosages, for youth with heterozygous familial hypercholesterolemia. Lovastatin, simvastatin, atorvastatin and fluvastatin are approved for children 10 years of age and older. Pitavastatin and pravastatin are approved starting at 8 years of age and rosuvastatin is indicated in children as early as age 6 (13-21). Table 3 provides a summary of HMG-CoA reductase inhibitors, pediatric approval and indications, recommended dosing ranges, comments on dosing, and supporting clinical trials (13-21).

Table 3. HMG-CoA Reductase Inhibitors
Medication	Pediatric Approvals & Indications	Dosing	Comments	Supporting Clinical Trials
Atorvastatin	Age 10-17 Heterozygous familial hypercholesterolemia	10-20 mg/day	May be titrated at ≥ 4-week intervals	McCrindle, et al (22)
Fluvastatin	Age 10-16 Heterozygous familial hypercholesterolemia	20-80 mg/day	May be titrated at ≥ 6-week intervals	van der Graaf, et al (23)
Lovastatin	Age 10-17 Heterozygous familial hypercholesterolemia	10-40 mg/day	Initiated at 20 mg/day for ≥20% LDL reduction, may be titrated at ≥ 4-week intervals	Clauss, et al (24) Lambert, et al (25) Stein, et al (26)
Pravastatin	Age 8 and older Heterozygous familial hypercholesterolemia	20-40 mg/day	Age 8-13: 20 mg/day Age 14-18: 40 mg/day	Knipscheer, et al (27) Wiegman, et al (28) Rodenburg, et al (29)
Rosuvastatin	Age 6 and older Heterozygous familial hypercholesterolemia	5-20 mg/day	May be titrated at ≥ 4-week intervals	Avis, et al (30)
Simvastatin	Age 10-17 Heterozygous familial hypercholesterolemia	10-40 mg/day	May be titrated at ≥ 4-week intervals	de Jongh, et al (31) de Jongh, et al (32)
Pitavastatin	Age 8 and older Heterozygous familial hypercholesterolemia	1-4 mg/day	May be titrated at ≥ 4-week intervals	Ferrari, et al (20)

The above are approved as an adjunct to a diet that is low in cholesterol and saturated fat. The above agents are approved for both males and females (females must be at least one-year post-menarche) if, despite an adequate diet and other non-pharmacologic measures, the following are present: LDL-C ≥ 190 mg/dL or LDL-C ≥ 160 mg/dL and the patient has a family history of premature cardiovascular disease or two or more cardiovascular disease risk factors.
Abbreviations: mg=milligrams, LDL=low density lipoprotein

As Table 4 outlines, statin therapies have demonstrated variable efficacy involving clinical trials of youth (21-32). With the longest half-life, rosuvastatin is the most potent statin, followed by atorvastatin (33). Given this knowledge, if pediatric patients who are initiated on statin therapy are having trouble meeting LDL-C goals, consideration should be given to switching to rosuvastatin or atorvastatin given potency prior to exploring second-line therapy options. Simvastatin is a moderately potent statin at clinically tolerable maximum doses of 40 mg/day (33-35). Lovastatin, pravastatin, and fluvastatin, respectively, are the least potent statins (34, 35). As many studies have demonstrated, reduced potency can be compensated by an increase in the amount of statin given; however, dose escalation is often associated with an increased occurrence of adverse events (21-35). As a result, selection of a specific statin therapy should be individualized and capable of reaching treatment goals. Equally important, consideration should be given to the prevalence and severity of reported side effects (36).

Table 4. Statin Therapy Results
Study	Medication	Dose	Results
Study	Medication	Dose	LDL-C	HDL-C	TC	TG
McCrindle, et al (22)	Atorvastatin	10-20 mg/day	-40%	+6%	-30%	-13%
van der Graaf, et al (23)	Fluvastatin	80 mg/day	-34%	+5%	-27%	-5%
Clauss, et al (24)	Lovastatin	40 mg/day	-27%	+3%	-22%	-23%
Lambert, et al (25)	Lovastatin	10 mg/day	-21%	+9%	-17%	-18%
Lambert, et al (25)	Lovastatin	20 mg/day	-24%	+2%	-19%	+9%
Lambert, et al (25)	Lovastatin	30 mg/day	-27%	+11%	-21%	+3%
Lambert, et al (25)	Lovastatin	40 mg/day	-36%	+3%	-29%	-9%
Stein, et al (26)	Lovastatin	10 mg/day	-17%	+4%	-13%	+4%
Stein, et al (26)	Lovastatin	20 mg/day	-24%	+4%	-19%	+8%
Stein, et al (26)	Lovastatin	40 mg/day	-27%	+5%	-21%	+6%
Knipscheer, et al (27)	Pravastatin	5 mg/day	-23%	+4%	-18%	+2%
Knipscheer, et al (27)	Pravastatin	10 mg/day	-24%	+6%	-17%	+7%
Knipscheer, et al (27)	Pravastatin	20 mg/day	-33%	+11%	-25%	+3%
Rodenburg, et al (29)	Pravastatin	20 mg/day or 40 mg/day	-29%	+3%	-23%	-2%
Wiegman, et al (28)	Pravastatin	20-40 mg/day	-24%	+6%	-19%	-17%
Avis, et al (30)	Rosuvastatin	5 mg/day	-38%	+4%	-30%	-13%
Avis, et al (30)	Rosuvastatin	10 mg/day	-45%	+10%	-34%	-15%
Avis, et al (30)	Rosuvastatin	20 mg/day	-50%	+9%	-39%	-16%
de Jongh, et al (31)	Simvastatin	10-40 mg/day	-41%	+3%	-31%	-9%
de Jongh, et al (32)	Simvastatin	40 mg/day	-40%	+5%	-30%	-17%

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report. Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

Although long term studies evaluating the safety and efficacy of lipid-lowering medications in youth are lacking, results of short term observational and randomized controlled trials are encouraging. For example, atorvastatin was found to be well tolerated with no statistically significant differences in adverse events reported for either the treatment or placebo groups (22). Additionally, the percentage of patients with abnormal laboratory results was similar for both groups; the only noted difference was an increased percentage of patients with elevated triglycerides in the placebo group. Treatment with atorvastatin resulted in no significant difference in sexual development as assessed by Tanner staging. Van der Graaf and colleagues found that in youth treated with fluvastatin, 58 (68.2%) reported non-serious adverse events. Only four were believed to be drug related. Treatment with fluvastatin resulted in no abnormalities in hormone levels or sexual maturation (23).

In their study of lovastatin, Clauss and colleagues reported no clinically significant alterations in vital signs; growth; hormone levels including luteinizing hormone, follicle-stimulating hormone, dehydroepiandosterone sulfate, estradiol, and cortisol; menstrual cycle length; liver function tests; or muscle function tests (24). Lambert and colleagues also found lovastatin generally well tolerated with no serious clinical adverse effects noted (25). While increased, levels of aspartate aminotransferase did not exceed two times the upper limit of normal and alanine aminotransferase did not display significant changes in study participants. Creatine kinase was elevated, greater than three times the upper limit of normal, in three patients. All subjects remained asymptomatic and the elevated creatine kinase levels resolved spontaneously while no adjustment was need in their medication. Assessment of growth and sexual maturation, by Tanner staging and estimation of testicular volumes, in youth treated with lovastatin found no significant differences between the treatment groups and placebo at either 24 or 48 weeks (26). While the authors reported no significant change in serum hormone levels or biochemical parameters of nutrition, they noted that the study was under powered to detect statistically significant changes in these safety parameters.

Use of pravastatin has been shown to have minimal adverse events dispersed evenly between the active drug recipients and those who received placebo (27). Plasma thyroid-stimulating hormone, adrenocorticotropic hormone, cortisol, creatine phosphokinase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, and alkaline phosphatase levels failed to show significant changes from baseline in all treatment groups. Rodenburg and colleagues also evaluated the safety of pravastatin based on annual or biannual evaluation of plasma creatine phosphokinase levels, liver enzymes, sex steroids, gonadotropins, and hormones of the pituitary-adrenal axis (29). Height, weight, age at menarche, Tanner staging, and testicular volume were recorded at baseline and either annually or biannually. Two subjects demonstrated elevated creatine phosphokinase levels which returned to normal without adjustments in therapy, and were presumed to be due to extreme physical exercise. No occurrence of myalgia was associated with elevation in levels of creatinine phosphokinase. None of the subjects discontinued therapy due to adverse events or laboratory abnormalities. Similarly, Wiegman and colleagues examined the safety of pravastatin evaluated at baseline, one year, and two years via multiple variables including: sex steroids, endocrine function parameters, height, weight, body surface area, Tanner staging, menarche or testicular volume, alanine aminotransferase, aspartate aminotransferase, and creatine phosphokinase (28). All safety parameters demonstrated no statistically significant differences between active drug recipients verses placebo in changes from baseline.

The safety of rosuvastatin was assessed by Avis and colleagues. Laboratory monitoring including liver enzymes and creatine kinase levels, and markers of growth and development, such as Tanner staging (30). Two serious adverse events were reported including blurred vision in one patient in the placebo group and a vesicular rash progressing to cellulitis in one patient taking rosuvastatin 20 mg. Transaminase levels either remained normal or normalized without permanent discontinuation of treatment. While elevations in creatine kinase and reports of myalgia did occur, symptoms and creatine kinase levels normalized without permanent discontinuation of therapy. Normal progression of height, weight, and sexual development were observed.

The safety of simvastatin during short term therapy has also been reported. Levels of alanine aminotransferase, aspartate aminotransferase, creatine kinase, and physical examination all demonstrated no significant differences between simvastatin treated and placebo participants (31). de Jongh and colleagues evaluated the safety of simvastatin in a second trial by monitoring adverse events as well as changes in alanine aminotransferase, aspartate aminotransferase, and creatine kinase levels (32). Of note, none of the differences reported in events or laboratory values between simvastatin recipients and placebo reached statistical significance. Additionally, there were no statistically significant differences documented for height, body mass index, cortisol levels, testicular size and testosterone levels, menstrual cycle and estradiol levels, and Tanner staging. Dehydroepiandrosterone sulfate levels demonstrated a statistically significant decrease in the simvastatin group compared to placebo.

Braamskamp and colleagues investigated the efficacy and safety of pitavastatin in pediatric patients diagnosed with hyperlipidemia. 106 patients were enrolled in the study, ages 6-17, for a 12-week period (37). Patients were randomly assigned to 4 different groups categorized by dose 1 mg, 2 mg, 4 mg, or placebo (37). The results showed a reduction in all 3 dose groups in comparison to placebo, the 1 mg group showed a 23.5% reduction in LDL-C, the 2 mg group showed a 30.1% reduction, and the 4 mg group showed a 39.3% reduction (37). There was also a 52-week extension period where patients assigned to the 1 mg group were up-titrated to a maximum dose of 4 mg to try and achieve an LDL-C level of >110 mg/dL (37). There were no safety issues of concern throughout the study. The results indicated that pitavastatin is safe and efficacious for use in pediatric patients, 6-17 years of age, and it was well-tolerated (37).

Long-term data regarding the impact of statin therapy on growth, development, and reduction of cardiac risk are limited, particularly for high intensity statin therapy. Recently, Kusters and colleagues evaluated the safety of statin therapy in children and adolescents with familial hypercholesterolemia after 10 years of treatment comparing laboratory safety markers as well as growth and maturation in untreated siblings (36). Only three patients discontinued therapy due to adverse events. Safety parameters such as aspartate aminotransferase, alanine aminotransferase, creatine kinase, estimated glomerular filtration rate, c-reactive protein, and age of menarche did not differ between treated patients and siblings, demonstrating safety over the 10-year treatment period. The authors do note; however, that the study was underpowered to detect the occurrence of rare events (36).

When initiating HMG-CoA reductase inhibitor therapy, as with any new medication therapy, it is imperative for clinicians to establish an accurate baseline, monitor for new symptoms, and counsel both patients and family members regarding potential adverse events. Females should be informed about the need to avoid pregnancy and breastfeeding while using statins. Statins may be taken with or without meals, but are commonly given with the evening meal or at bedtime as this has the potential to improve LDL-C reduction (38). As a major substrate of P450, such as CYP3A4, there are multiple drug interactions associated with statin therapy. Grapefruit juice has gained considerable notoriety as a potential food interaction; however, it should be noted that more than a quart of grapefruit juice would have to be consumed to increase serum statin levels. Of more concern is the possible interaction of gemfibrozil and statins, which should either be avoided as it can increase the toxicity of HMG-CoA reductase inhibitors. Macrolides and antifungal azoles are classes of drugs commonly prescribed to children, and they should be avoided as often as possible as they increase serum statin levels leading to potentially enhanced myopathic effects. Additionally, patients should avoid herbal products and nutraceuticals, such as red yeast rice, which may further enhance adverse effects.

BILE ACID SEQUESTRANTS

Bile acid sequestrants, or bile acid binding resins, present an additional treatment option for youth with severe dyslipidemia. Bile acid sequestrants represent one of the oldest classes of medications available to treat dyslipidemia and were the only medication recommended in the 1992 NCEP Pediatric Panel Report, at a time when no data were available for statin use in youth (4). While no longer a recommended first-line therapy, bile acid sequestrants do have potential as a treatment option either alone or in combination with a statin (9, 10). At present, colesevelam is the only bile acid sequestrant with FDA approval for youth age 10 years and older with heterozygous familial hypercholesterolemia (39, 40). Despite the lack of FDA approval, both colestipol and cholestyramine have been studied in pediatric patients (41-49).

A number of clinical trials have evaluated the bile acid sequestrants in pediatric patients with heterozygous familial hypercholesterolemia and other forms of severe dyslipidemia. While palatability and tolerance remain potential barriers to effective therapy, in general, bile acid sequestrants have demonstrate significant reductions in both total cholesterol and LDL-cholesterol in study subjects (40, 43, 44, 48, 49). Table 5 provides a summary of bile acid sequestrants, pediatric approval and indications, recommended dosing ranges, comments on therapy, and supporting clinical trials. As outlined in Table 6, studies demonstrated some variability in efficacy for the available bile acid sequestrants (40, 43, 44, 48, 49).

Table 5. Bile Acid Sequestrants
Medication	Pediatric Approvals & Indications	Dosing	Comments	Clinical Trials
Colesevelam	Age 10-17 Heterozygous familial hypercholesterolemia	1.875 g twice daily or 3.75 g daily	May be used as monotherapy or in combination with a statin	Stein, et al (40)
Colestipol	(Note: Not FDA Approved) Age 7-12 Primary hypercholesterolemia	5 g twice daily, 10 g daily, or 125-500 mg/kg/day	N/A	McCrindle, et al (43) Tonstad, et al (44)
Colestipol	(Note: Not FDA Approved) Age ≥12 Primary hypercholesterolemia	10-15 g/day	N/A	McCrindle, et al (43) Tonstad, et al (44)
Cholestyramine	(Note: Not FDA Approved) Age 6-12 Hypercholesterolemia adjunct	240 mg/kg/day divided three times daily before meals	Initiate at 2-4 g twice daily	McCrindle, et al (48) Tonstad, et al (49)
Cholestyramine	(Note: Not FDA Approved) Age ≥12	8 g/day divided twice daily before meals	N/A	McCrindle, et al (48) Tonstad, et al (49)

Colesevelam is approved as an adjunct to a diet that is low in cholesterol and saturated fat. Colesevelam is approved for both males and females (females must be at least one-year post-menarche) if, despite an adequate diet and other non-pharmacologic measures, the following are present: LDL-C ≥ 190 mg/dL or LDL-C ≥ 160 mg/dL and the patient has a family history of premature cardiovascular disease or two or more cardiovascular disease risk factors.
Abbreviations: g=grams, mg=milligrams, kg=kilograms, N/A=not applicable.

Table 6. Bile Acid Sequestrant Results
Study	Medication	Dose	Results
Study	Medication	Dose	LDL-C	HDL-C	TC	TG
Stein, et al (40)	Colesevelam	1.875 g/day	-6%	+5%	-3%	+6%
Stein, et al (40)	Colesevelam	3.75 g/day	-13%	+8%	-7%	+5%
McCrindle, et al (43)	Colestipol	10 g/day	-10%	+2%	-7%	+12%
McCrindle, et al (43)	Colestipol & Pravastatin	Colestipol: 5 g/day Pravastatin: 10 mg/day	-17%	+4%	-13%	+8%
Tonstad, et al (44)	Colestipol	2-12 g/day	-20%	-7%	-17%	-13%
McCrindle, et al (48)	Cholestyramine	8 g/day	-10% to -15%	+2% to +4%	-7% to -11%	+6% to +9%
Tonstad, et al (49)	Cholestyramine	8 g/day	-17%	+8%	-12%	N/A

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report. Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: g=grams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

Stein and colleagues assessed the safety of colesevelam at weeks 8-26 during an open-label study from. All subjects received colesevelam 3.75 grams per day in addition to a statin (40). Safety was measured via adverse events, vital signs and physical exam, laboratory monitoring, and Tanner staging. The most common adverse events related to use of colesevelam were gastrointestinal, including diarrhea, nausea, vomiting, and abdominal pain. It is important to note that no choking or difficulty swallowing were reported with the use of colesevelam. Vital signs, physical exams, laboratory monitoring, and Tanner staging remained the same or progressed as expected throughout the study period.

McCrindle and colleagues evaluated conventional high-dose colestipol versus a combination of low-dose colestipol plus pravastatin, but did not cite safety as an endpoint for their study (40). The researchers did conduct safety monitoring in the form of laboratory tests, physical evaluation, and adverse event reporting. Significant deviations from baseline were noted for alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase at various time intervals with different medication regimens; however, the authors noted that when compared to reference values, none of the laboratory results were considered abnormal. Study participants in the two medication regimens did not significantly vary in weight gain, height changes, or body mass index. While the majority of patients experienced no adverse events, gastrointestinal symptoms such as constipation, gas or bloating, or stomach ache were more commonly reported by the patients taking the high-dose colestipol. The authors found similar suboptimal compliance with both regimens as determined by medication counts at the end of each study period. Tonstad and colleagues also assessed the tolerability of colestipol granules by monitoring side effects as well as by having subjects’ complete subjective evaluations (44). Side effects associated with colestipol included constipation, dyspepsia, flatulence, nausea, reduction in appetite, and abdominal pain. The subjective evaluations indicated that only 21% of patients liked the taste of the colestipol; however, of those who had previously taken bile acid binding resin, 86% preferred the taste of the newer orange flavored granules. Thirty seven percent of subjects also reported that they frequently forgot to take the medication, while 44% intentionally eliminated the medication from their routine on special occasions or during trips.

The acceptability and compliance of cholestyramine has been studied (48). Eighty two percent (82%) of children preferred the pill formulation of cholestyramine compared to 16% who preferred the powder. Two percent of children in the study preferred neither form of the medication. Compliance was significantly impacted by medication formulation with patients taking the pill form reporting 61% compliance while those on the powder formulation were only 50% compliant. Compliance increased by at least 25% for 42% of patients when they switched to the pill formulation. Tonstad and colleagues assessed the safety of cholestyramine by measuring height velocity, erythrocyte folate, total plasma homocysteine, serum fat-soluble vitamins, and side effects (49). Weight and mean height velocity standard deviation scores were not statistically significant between treatment and placebo groups during the study. The cholestyramine active treatment group demonstrated decreased vitamin D levels and increased homocysteine levels. Differences in erythrocyte folate were not significant between the active treatment and placebo groups. Reported adverse events included intestinal obstruction, abdominal pain, nausea, and loose stools. Unpalatability was a common reason participants withdrew from the study.

As demonstrated by the previous studies, while bile acid sequestrants do present an effective therapy option, their side effect profile, issues of tolerability and drug interactions with statins make their use clinically challenging. It is generally recommended that all concurrent medications be given either one hour before or four hours after bile acid sequestrants to prevent decreased absorption of the additional therapies (41, 46). Use of bile acid sequestrants is generally limited to patients optimized on statin therapy who require additional therapy to achieve goal or those that cannot tolerate statins. Data on long-term safety, however, are generally lacking. It should also be noted that bile acid sequestrants can increase triglyceride levels and should not be used in patients with increased triglyceride levels.

FIBRIC ACID DERIVATIVES

Experience with fibric acid derivatives in youth is limited. Currently there are no fibric acid derivatives with FDA approval for use in pediatric patients. Both fenofibrate and gemfibrozil are available in the United Sates, but lack pediatric data on safety, efficacy, and dosing (50, 51). While there is very limited information on the use of bezafibrate in youth, the product is not available in the United States (52). It should be noted that fibric acid derivatives have the potential to increase the incidence in adverse events, such as rhabdomyolysis, when used with statins (45, 50, 51). However, the use of fibrates should be considered and can be beneficial in pediatric patients who also have triglyceride abnormalities (TG levels > 500 mg/dL) (9, 53).

NIACIN

Niacin provides a potential adjunct therapeutic option for youth with severe dyslipidemia who have not achieved their lipid goal. Extended-release niacin is the only formulation that has FDA-approval for use in children > 16 years of age (54). Despite a lack of FDA approval for ages younger than 16, limited efficacy and safety data are published for the use of niacin in children 10 years of age as older as adjunct therapy (54). Table 7 summarizes data on recommended dosing ranges, comments on dose adjustments, and references supporting clinical trials.

Colletti and colleagues conducted a retrospective review to evaluate the efficacy and adverse effect profile of niacin for children with severe hypercholesterolemia (54). The effects on serum lipid profiles are detailed in Table 8. Adverse effects were common, affecting 76% of children, and similar to those reported for adults including: flushing, abdominal pain, vomiting, headache, and elevated liver enzymes. Due to the high prevalence of adverse effects, use of niacin should be limited to patients not achieving goal with other therapies or those who cannot tolerate alternative adjunctive options. As with fibrates, niacin can also be considered for the purposes of treating pediatric patients who are concurrently diagnosed with hypertriglyceridemia (9).

Table 7. Niacin

Medication

Pediatric Approvals & Indications

Dosing

Comments

Clinical Trials

Niacin

Extended release: >16 years of age

Note: Immediate release is not FDA Approved:
Age ≥ 10
Adjunct therapy

Initial: 100-250 mg/day
(Max: 10 mg/kg/day)
divided three times daily with meals

May titrate weekly by 100 mg/day or every 2-3 weeks by 250 mg/day

Colletti, et al (54)

Abbreviations: mg=milligrams, kg=kilograms.

Table 8. Niacin
Study	Medication	Dose	Results
Colletti, et al (54)	Niacin	500-2,250 mg/day	LDL-C	HDL-C	TC	TG
Colletti, et al (54)	Niacin	500-2,250 mg/day	-17%	+4%	-13%	+13%

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report. Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides.

EZETIMIBE

Ezetimibe is FDA approved for adolescents 10 years of age and older with FH (53, 55), and it presents a potential therapy option either as monotherapy or when synergistically paired with an HMG-CoA reductase inhibitor (56-58). Due to its favorable tolerability, it has become the most frequently used second-line agent (59). Table 9 summarizes data on recommended dosing ranges and references supporting clinical trials while Table 10 details efficacy of therapy.

Tolerability of ezetimibe was prospectively evaluated by Yeste and colleagues via a combination of biochemical markers and adverse event reports (56). No change was seen in hemogram, transaminases, creatinine, calcium, phosphorus, and vitamins A and E for any of the 17 patients. Additionally, there were no reports of adverse events during the study period. Clauss and colleagues retrospectively evaluated ezetimibe; therefore, safety parameters were less defined, but included intermittent measurement of liver enzymes, occasional CK levels, and adverse event reports (57). There were no reported abnormalities in liver enzymes for study participants. Ultimately, one patient was discontinued from ezetimibe therapy for asymptomatic elevated CK levels, later determined to be likely unrelated to therapy.

Table 9. Ezetimibe
Medication	Pediatric Approvals & Indications	Dosing	Comments	Clinical Trials
Ezetimibe	Age ≥10 Homozygous familial hypercholesterolemia	10 mg/day	N/A	Yeste, et al (56) Clauss, et al (57) van der Graaf, et al (58)

Abbreviations: mg=milligrams, N/A=not applicable.

Table 10. Ezetimibe
Study	Medication	Dose	Results
Yeste, et al (56)	Ezetimibe	10 mg/day	LDL-C			HDL-C	TC	TG
			PH		-42%	N/A	-31%	N/A
			FH		-30%	-15%	-26%	N/A
Clauss, et al (57)	Ezetimibe	10 mg/day	LDL-C			HDL-C	TC	TG
			FH	-28%		N/A	-22%	N/A
			FCHL	-28%		N/A	-13%	N/A
van der Graaf, et al (58)	Ezetimibe & Simvastatin	Ezetimibe: 10 mg/day Simvastatin: 10-40 mg/day	LDL-C			HDL-C	TC	TG
van der Graaf, et al (58)	Ezetimibe & Simvastatin	Ezetimibe: 10 mg/day Simvastatin: 10-40 mg/day	-49%			+7%	-38%	-17%

Adapted from National Heart Lung and Blood Institute (NHLBI): Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary Report. Pediatrics. 2011;128(S5):S213-S256: Table 9-11.
Abbreviations: mg=milligrams, LDL-C=low density lipoprotein cholesterol, HDL-C=high density lipoprotein cholesterol, TC=total cholesterol, TG=triglycerides, PH=polygenic hypercholesterolemia, FH=familial hypercholesterolemia, FCHL=familial combined hyperlipidemia.

Van der Graaf and colleagues assessed the safety of combination therapy with ezetimibe and simvastatin based on reported adverse events as well as laboratory monitoring and clinical examination (58). After 53 weeks, 71% of study participants reported some types of treatment-emergent adverse events. Of those events reported, only influenza, nasopharyngitis, and headache occurred in greater than 5% of participants. Consecutive transaminase elevations of at least three times the upper limit of normal were reported in 6 participants; however, all values resolved with interruption or discontinuation of therapy. Elevations in creatine phosphokinase occurred infrequently and were not associated with myalgia at levels greater than three times the upper limit of normal. Height, weight, and sexual maturation were not significantly impacted by therapy. Ezetimibe affords flexibility in administration time with the ability to administer it without regard to meals or time of day (45). HMG-CoA reductase inhibitors have the risk of increasing myopathy and elevation in hepatic transaminases, but are generally considered a safe combination with ezetimibe.

OMEGA-3 FISH OILS

Omega-3 fish oils are a class of therapy for which there is significantly limited data in youth. To date the FDA approved formulations of omega-3 fatty acid lack a pediatric indication. High dose omega-3 fatty acid supplementation was evaluated by de Ferranti and colleagues, but ultimately the authors found no statistically significant improvement when subjects were compared to placebo (60). Chahal and colleagues similarly found no significant impact on hypertriglyceridemia when treating pediatric patients with fish oil (61).

Khorshidi and associates performed a systematic review and meta-analysis of the effect of omega-3 supplementation on lipid profiles in children and adolescents. They found that omega-3 supplementation improved triglyceride levels in patients diagnosed with hypertriglyceridemia that were less than or equal to 13 years of age; however, there was no significant effect seen in HDL, LDL, or TC values (62). Omega-3 fish oils can be considered in those who have elevated triglyceride levels.

FAMILIAL HYPERCHOLESTEROLEMIA AND THERAPEUTIC ADVANCES

Familial hypercholesterolemia (FH) is a common, but often misdiagnosed inherited gene disorder (63). The most common gene mutation seen in FH is in the low-density lipoprotein receptor gene (LDLR) accounting for 85-90% of cases, followed by the apolipoprotein (ApoB) gene (5-15% of cases) and the proprotein convertase subtilisin kexin 9 (PCSK9) genes (1% of cases) (63, 64). Patients who are diagnosed with FH have abnormally elevated LDL levels from birth. FH is associated with a twenty-fold increased risk in premature cardiovascular disease and cardiovascular events (65). There are two different types of FH, heterozygous (HeFH) and homozygous (HoFH). Heterozygous patients have one mutated allele and are more commonly seen in practice, while homozygous patients have two mutated alleles and are very rare (66). Distinction between the two types is imperative because HoFH patients tend to be treatment resistant and carry a worse prognosis, if left untreated, these patients rarely make it past age 20 (66).

Lipid lowering in these patients can be quite challenging, especially HoFH patients. Most typical therapies for lipid lowering require functional LDL receptors, therefore given the gene mutations which often render the receptors inactive, modest reductions of 10-25% in HoFH patients are usually all that will be gained (67). HeFH patients tend to see higher rates of reduction (25-40%) (68). However, first line treatment for both forms of FH is still high-intensity statin therapy at moderate to high doses to be initiated as early as age 8 (69). All seven statins are FDA-approved for the treatment of FH and have been proven to slow down the progression of carotid intima-media thickness (63). Statins also reduce the incidence of cardiovascular events and cardiac death (63). Until recently, long term data on the use of statins in this patient population was not available, but in 2019, a 20-year follow-up study of statins in pediatric patients with FH was published (70). The results found that the incidence of cardiovascular events and death was much lower in patients treated with statins (70). If LDL-C goals are not being met with the use of statins alone, the next recommend agent is ezetimibe; however, this should not be used in patients younger than 10 years of age (59).

Ezetimibe is the second-line option to statins in these patients. It’s use in combination with statins has demonstrated a reduction of LDL-C levels below 135 in more than 90% of children with FH (71). In one of the only studies that assessed the coadministration of simvastatin and ezetimibe in children with HeFH, it was found to be safe, well tolerated and provided a higher LDL reduction (15%) compared with simvastatin alone in HeFH patients (72). However, when it was investigated as a monotherapy option for children with HeFH, it only produced LDL-C lowering of 27% (71, 73). It is more appropriate to use as an adjunct therapy in this population of patients.

Alternative medications that can be considered other second-line options are bile acid sequestrants (71). Colesevelam is safe to use, but limited to children >10 years of age (71). These drugs however have minimal LDL-C lowering effects, usually only seeing a 10-20% reduction, and more importantly are very poorly tolerated due to gastrointestinal side effects (71).

PCSK9 Inhibitors

Given the difficulty and importance of treating these patients, especially those with HoFH, there is a need for stronger lipid lowering options, which is where PCSK9 inhibitors come into play. These are a more recent class addition to the therapy options for managing FH, which help to reduce the degradation of LDL receptors and the removal of LDL-cholesterol (74). In a recent study assessing the efficacy/safety of lipid-lowering agents in patients with familial hypercholesterolemia, it was concluded that PCSK9 inhibitors were the most effective in lowering lipid levels (75). They have none of the same side effects as statins and produced similar CV benefits. Therefore, based on these conclusions, PCSK9 inhibitors are recommended as first-line agents in patients with hypercholesteremia that have intolerances or resistance to statins (75). There is currently one PCSK9 inhibitor approved for pediatric use. Repatha (evolocumab) was originally approved for use in patients 13 years of age and older (76), but the HAUSER-RCT study assessed the use of Repatha in patients ages 10-17 years of age for 24 weeks. It showed that the drug improved lipid levels (by approximately 38% in HeFH patients and 21-24% in HoFH patients) and was safe for use as the incidence of adverse events was similar in both the drug and placebo groups (71, 77). So now it is considered safe to use in pediatric patients 10 years of age and older. The HAUSER-RCT trial was then continued for another 80 weeks to further assess the safety and efficacy of Repatha (78). This new trial, HAUSER-OLE, further confirmed that the drug was safe and well-tolerated (78).

Praluent (Alirocumab) is another PCSK9 inhibitor that is available for treatment of FH, however it is not currently approved for use in pediatric patients (79). Nevertheless, there are studies currently assessing the safety and efficacy within this population. Bruckert and associates utilized Praluent and conducted an open-label phase 3 study specifically in pediatric patients (8-17 years of age) diagnosed with HoFH that were inadequately controlled (80). Patients received 75 or 150 mg of the drug based on weight (<50 or >50 kg, respectively) every 2 weeks for 12 weeks (80). The primary endpoint was percent change in LDL-C levels from week 0 to 12 (80). Interestingly, the results showed only a 4.1% decrease in LDL-C levels by week 12 (80). The secondary endpoints (assessing percent change LDL-C levels from baseline to weeks 24 and 48, changes in other lipid parameters from baseline to weeks 12, 24, 48, patients with a reduction of more than 15% in LDL-C levels at weeks 12, 24 and 48, and absolute change in LDL-C from baseline to weeks 12, 24, and 48) produced incredibly variable results (80). Overall, there were quite small changes in LDL-C levels observed in this study with mean reductions of LDL-C levels noted to range anywhere from ~33 to 52 mg/dL (80). More importantly, previous studies have shown that PCSK9 inhibitors are linked to a decrease in major coronary/vascular events and all-cause mortality, so although the results produced small values, the changes seen are still clinically significant based on these added benefits (80). It was also noted that this study produced similar results when compared to the ODYSSEY study which assessed the use of Praluent in adult patients with HoFH (80). The drug was deemed safe and there were no issues with tolerability (80). The study supports the use of Praluent as an adjunct therapy in HoFH patients already on first- and second-line therapies and not reaching their goal LDL-C levels (80).

Another study assessed the use of Praluent in pediatric patients diagnosed with HeFH. The ODYSSEY KIDS study was a phase 2 dose-finding study that enrolled pediatric patients anywhere from 8-17 years of age (81). Patients were split into 4 cohorts and dosed every 2 weeks. Dosing was determined by weight and the primary endpoint assessed percent change in LDL-C from baseline to week 8 (81). Praluent demonstrated the best reduction in LDL-C levels in the highest dosed cohorts and was well-tolerated. This study also supported the use of the drug (with further analysis) in patients who require adjunct therapy, there is a phase 3 trial planned to assess the doses from this study that resulted in the greatest reduction in LDL-C levels. Overall, it is important to note that HoFH patients are more likely to fail PCKS9 inhibitors (82). This is attributable to their mechanism of action. This class of medication requires functional LDL receptors, and this is impaired or completely absent in HOFH patients (82). Therefore, effectiveness of PCSK9 inhibitors tends to be much higher in HeFH patients (82).

Leqvio (inclisiran) is also another PCSK9 inhibitor currently not approved for pediatric use (83). The mechanism of action of this drug differs from Repatha and Praluent. Leqvio is a small interfering RNA (siRNA) that utilizes the RNA interference mechanism to cause the catalytic breakdown of mRNA for PCSK9, thus stopping the translation of the protein (84). It also only requires administration twice yearly as opposed to biweekly (84). There are currently ongoing studies investigating the possibility of using Leqvio in pediatric patients. ORION-13 and ORION-16 are studies assessing the efficacy, safety and tolerability of Leqvio in pediatric patients diagnosed with HoFH and HeFH, respectively (84). They are two-part (1-year double blind, the other year open-label) phase 3 trials consisting of patients aged 12 to <18 years with FH (84). The primary endpoint is the percentage change in LDL-C from baseline to day 330 (84). Based on the results, this could be another drug option as adjunct therapy to consider for use.

Angiopoietin-Like Protein 3 (ANGPTL3)

Angiopoietin-like protein 3 (ANGPTL3) also presents a novel target of adjunctive therapy for patients with homozygous familial hypercholesterolemia that are not meeting LDL-C goals with first-line agents (85, 86). Evkeeva (evinacumab-dgnb) is a monoclonal ANGPTL3 inhibitor that is FDA-approved specifically for the adjunctive treatment of homozygous familial hypercholesterolemia in patients 5 years of age and older (87).

Lomitapide

Lomitapide is another potential treatment option for patients with HoFH. This medication works differently from more conventional options. It binds to microsomal triglyceride transfer protein (MTP) and prevents the production of lipoproteins that contain apo-B (88). This causes a decrease in the production of very-low-density lipoprotein (VLDL) and chylomicrons. Since VLDL is converted into LDL, this mechanism ultimately causing a decrease in LDL-C levels (89). It is administered once daily at doses ranging from 5 to 60 mg (88). The side effect profile of lomitapide can be difficult for patients as it can cause severe gastrointestinal side effects (due to the decrease in absorption of fats in the intestines), most often diarrhea (89). But it is also associated with raised hepatic fats and enzymes (82). It is currently approved for adult use only, but it has become an option for use in pediatric patients through an expanded access program or a named patient basis (82). There was a case series done exploring the effect of lomitapide in 11 pediatric patients diagnosed with HoFH. It demonstrated that the drug was effective in reducing LDL-C with all 11 HoFH patients and showed a similar side effect profile to that seen in adult patients (82). GI complaints were moderated and did not cause any discontinuation of use (82). It also showed greater reduction in LDL-C levels at lower doses (82). The greatest benefit of lomitapide was associated with its ability to reduce or stop the need for lipoprotein apheresis in the patients incorporated in this case study (82). An interesting mention about lomitapide from the case series is that adult patient data shows that early intervention utilizing the drug showed a potential for increased life expectancy and a delay in the time to first major adverse cardiovascular event (82). There is also currently an ongoing phase 3, open label trial investigating the efficacy and safety of lomitapide in pediatric patients with HoFH, estimated completion date is April of 2024 (82).

Bempedoic Acid

Bempedoic acid is a new medication that exerts its effects very similarly to that of statins. It works in the same pathway as statins and targets cholesterol biosynthesis (90-92). It is administered however as a prodrug and converted to active drug only in the liver and not in the muscles (90-92). The other difference between the two classes is that bempedoic acid inhibits ATP-citrate lyase (ACL), while statins inhibit HMG CoA reductase (90-92). Due to the lack of activation in skeletal muscles, this drug is a promising alternative to patients unable to take statins due to muscle related symptoms (90-92). The medication is FDA approved for use in patients with HeFH and those with established cardiovascular disease (93). It has shown promising results in adult trials, but there are currently no published pediatric trials to date assessing the safety or efficacy of use of the drug (93). There does however appear to be a trial in development: “An Open-Label Study to Evaluate the Pharmacokinetics, Pharmacodynamics, and Safety of Bempedoic acid in Pediatric Patients with Heterozygous Familial Hypercholesterolemia.” The results are highly anticipated so that this can offer another promising drug class for use in patients intolerant or unable to meet their LDL-C goals.

FAMILIAL CHYLOMICRONEMIA SYNDROME

Familial chylomicronemia syndrome (FCS) is an incredibly rare autosomal recessive gene disorder (94). There is reduced or absent lipoprotein lipase activity causing disruption in chylomicron metabolism leading to severely elevated triglyceride levels resulting in acute recurrent pancreatitis (94). There is not however an increased risk of ASCVD with an FCS diagnosis (94). The best way to treat FCS is also often referred to as the most difficult as it requires patients to restrict dietary intake to <10-15% of daily calories (94). Other treatment options utilized are fibrates, omega-3 fatty acids and statins with variable responses, but the use of these medications is most commonly seen in patients who have multifactorial chylomicronemia syndrome (94). Given the difficulty of ensuring these patients maintain low levels of triglycerides, medications like volanesorsen are being examined (94, 95).

Volanesorsen

Volanesorsen is a second-generation 2’-O-methoxyethyl (2’-MOE) antisense therapeutic oligonucleotide. It works by inhibiting apoC3 thus lowering triglyceride plasma levels (94). When it binds to apoC3, this interrupts mRNA translation which consequently promotes triglyceride clearance/lowering of triglyceride plasma levels (94). The efficacy and safety of volanesorsen was assessed in the APPROACH study (96). It included 67 patients that were randomized to either weekly volanesorsen or placebo for 3 months (97). The results showed a 77% reduction in triglyceride plasma levels at the end of the study period and there was only 1 event of pancreatitis in the study group (97). The largest trial performed assessing the use of volanesorsen was the COMPASS trial (97). It included 114 patients who were randomized to either weekly injections of volanesorsen or placebo for a total of 26 weeks (97). The results showed that patients in the treatment group saw a reduction in triglyceride levels, chylomicron triglycerides, VLDL levels and apoC3 levels by more than 70% (97). There were also no occurrences reported of pancreatitis in any of the patients randomized to the volanesorsen group (93). In both trials, volanesorsen proved itself as a promising agent for treatment of hypertriglyceridemia in FCS patients (95-98). This drug is not approved for use in the US but is approved in other countries.

CONCLUSION

As noted in the 2011 NHLBI’s guidelines, available information regarding the treatment of youth with lipid disorders has greatly expanded. HMG-CoA reductase inhibitors, or statins, are now considered first-line pharmacologic treatment of children and adolescents with severe hypercholesterolemia who fail treatment with diet and exercise alone, although statins are only FDA approved for youth with familial hypercholesterolemia. Despite their ability to effectively reduced cholesterol levels, use of bile acid sequestrants continue to pose challenges for pediatric patients due to their unpalatability and are typically utilized as adjunctive therapy or for patients not able to tolerate statins. Fibric acid derivatives, as a class of medications, not only lack an FDA approved agent, but also continue to lack significant pediatric safety and efficacy data. Niacin, a potential adjunct therapy, lacks FDA approval for pediatric patients and is plagued by significant adverse effects, making it an unlikely therapeutic option for youth. Ezetimibe provides clinicians with an alternative adjunct therapy option when synergistically paired with an HMG-CoA reductase inhibitor or used as monotherapy for patients intolerant to statins and bile acid sequestrants. Despite their inherit appeal and popularity amongst the lay public, omega-3 fish oils have failed to demonstrate statistically significant cholesterol lowering in pediatric and adolescent patients, but can be used to lower triglyceride levels. PCSK9 and ANGPTL3 inhibiting agents are promising novel treatment options in pediatric patients diagnosed with FH. While recent years have witnessed a dramatic increase in studies of lipid lowering medications in youth, the long-term safety and efficacy data continue to present an active focus of research.

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Ambiguous Genitalia in the Newborn

ABSTRACT

Ambiguous genitalia in a newborn are the clinical sign of atypical sexual development of the external genitalia in utero. This condition is rare and can result from various underlying factors, including certain disorders with potentially severe consequences, such as cortisol deficiency due to congenital adrenal hyperplasia. Therefore, it is crucial to promptly determine etiology when ambiguity is observed. The formation of typical male or female external genitalia is a complex process involving a cascade of genetic and physiological events that begin with sex determination and progress through the differentiation of internal and external reproductive structures. When this process is disrupted and does not occur in the typical manner, it is referred to as a difference or disorder of sex development (DSD). Not all DSD cases present with ambiguous genitalia at birth; for example, complete androgen insensitivity syndrome does not, but all cases of ambiguous genitalia are the result of a DSD. This chapter focuses on genital ambiguity associated with DSD in newborns who have either a 46,XY or 46,XX chromosomal sex. However, DSD with genital ambiguity may also be observed in newborns with other combinations of sex chromosomes, such as 45,X/46,XY. The chapter offers a comprehensive overview of the evaluation and management of newborns with ambiguous genitalia. It emphasizes the importance of a structured medical assessment of the external genitalia to diagnose and determine the underlying cause of genital ambiguity. It includes tables for differential diagnosis and step-by-step workup algorithms to guide medical professionals in their evaluation. It also includes additional information on structured physical assessments of external genitalia and tables with normative values for hormonal measurements, which are recommended in the diagnostic process. As the etiology of genital ambiguity in newborns is diverse and can have significant implications for management, the authors stress that obtaining an accurate diagnosis through a professional medical workup is crucial. The chapter highlights the recommendation for newborns with ambiguous genitalia (DSD) to be cared for by highly specialized, interdisciplinary DSD teams. These teams are equipped with medical and psychosocial expertise, and specialized psychologists are available to support parents and caregivers. The chapter recognizes that having a child with ambiguous genitalia can be very stressful for parents. It underscores the importance of early education, access to expert care through DSD network teams, psychological support, shared decision-making, and promoting acceptance and inclusivity. By providing comprehensive support and guidance, families can better navigate the challenges and uncertainties they may encounter when caring for a newborn with ambiguous genitalia.

INTRODUCTION

Typical male and female development commences with the presence of the typical 46,XY or 46,XX chromosomes, which play a pivotal role in determining the indifferent gonads. Around the 6th week of gestation, these gonads follow pathways to develop into male-typical or female-typical gonads (Figure 1). This gonadal determination is intricately regulated by a complex interplay of multiple genes, which guide the male gonads to become testes and the female gonads to become ovaries.

Subsequently, the gonads initiate the production of various hormones, including anti-Müllerian hormone (AMH) and testosterone. These hormones are responsible for driving the differentiation of the embryologic Wolffian and Müllerian structures into sex-typical internal reproductive organs (Figure 1).

Figure 1. Typical male and female sex determination and differentiation occur during fetal life. The presence of the Y chromosome in the karyotype and the SRY gene (sex-determining region Y gene) are the primary determinants of male sex development. The SRY gene initiates a cascade that leads to the development of testes from bipotential gonads. Testosterone and Anti Mullerian Hormone (AMH) are secreted by testicular cells, which masculinize the Wolffian (mesonephric) duct and cause the regression of the Mullerian (paramesonephric) duct, respectively. Dihydrotestosterone (DHT), produced by the 5-alpha reduction of testosterone, plays a major role in male external genital development and prostate formation. The absence of the SRY gene on the Y chromosome is the primary factor for the induction of the female sex, and the development of the uterus, fallopian tubes, cervix and upper vagina from the Mullerian duct.

The development of external genitalia begins with a neutral anlage, including a genital tubercule, genital folds, and a urogenital sinus. These structures are subsequently differentiated into typical male external genitalia under the influence of androgens, particularly dihydrotestosterone (DHT). Conversely, for the development of typical female external genitalia, the absence of androgens is crucial to prevent virilization.

Therefore, ambiguous genitalia at birth can arise from either an excessive or insufficient androgen effect on the neutral external genital anlage in males or females, respectively (Figure 2).

Figure 2. External genital differentiation. The development of external genitalia begins with a bipotential anlage which includes genital tubercle, urethral fold, urethral groove, and genital swellings. In a typical 46, XY, androgen secretion from the testes triggers the fusion of urethral folds, allowing the enclosure of the urethral tube. This, together with the cells from the genital swelling, forms the shaft of the penis. Genital swellings fuse in the midline to allow formation of the scrotum and genital Tubercle expands to give rise to the glans penis. Female external genital development is ensured by the absence of testosterone, it is independent of ovarian endocrine activity. Urethral folds and genital swellings remain separate to form the labia minora and majora. Genital tubercle forms the clitoris.

CHARACTERISTICS OF TYPICAL MALE AND FEMALE EXTERNAL GENITALIA

A full-term male infant is typically expected to have bilateral testicles that are descended, complete formation of scrotal folds with midline fusion, and a typical-sized penis, which includes well-formed corporal bodies and a urethral meatus located at the tip. The average penile length for a full-term newborn is approximately 3.5 ± 0.4 cm, but this measurement can vary with gestational age (Table 1) (1, 2). If an infant presents with bilateral cryptorchidism, a bifid scrotum, hypospadias, or isolated penoscrotal hypospadias, further investigations for a DSD are recommended. However, it’s important to note that isolated micropenis, as long as both testes are descended and normal in size, is not typically considered a manifestation of ambiguous genitalia. Similarly, in males, distal hypospadias with no other atypical genital features usually does not indicate a DSD (3).

Table 1. Stretched Penile Length in Preterm and Term Newborns According to Gestational Age (2)
Gestational age (week)	Number of cases	Median (cm)	Mean (cm)	SD	Min (cm)	Max (cm)
26	30	1.9	1.9	0.32	1.1	2.5
27	31	2.1	2.0	0.28	1.1	2.6
28	32	2.0	1.9	0.31	1.3	2.9
29	29	2.3	2.3	0.39	1.3	2.9
30	31	2.4	2.4	0.32	1.6	3.0
31	33	2.4	2.4	0.38	1.8	3.3
32	32	2.9	2.6	0.44	2.0	3.2
33	28	2.9	2.8	0.45	1.9	3.4
34	30	2.9	2.8	0.43	2.0	3.6
35	29	3.1	2.9	0.61	2.0	4.1
36	31	3.1	3.0	0.50	2.2	4.0
37	29	3.1	3.0	0.47	2.2	4.1
38	44	3.1	3.1	0.54	2.1	4.5
39	82	3.2	3.2	0.55	2.0	4.2
40	65	3.5	3.4	0.53	2.2	4.4
41	29	3.6	3.5	0.47	2.6	4.3

A full-term female infant typically presents with bilateral separated labial folds, no palpable gonads, and distinct urethral and vaginal openings (Figure 2). The average clitoral length and width for a full-term infant girl are approximately 6.1 mm and 4.2 mm, respectively (1, 4). In cases where clitoromegaly (clitoral size ˃10 mm), labial fusion or palpable gonads are observed in otherwise typical female genitalia, further investigation for a DSD is warranted. It’s important to note that perceived clitoromegaly is not typically associated with an underlying DSD in the event that the newborn girl was born prematurely (1). Please be aware that perceived clitoromegaly or pseudo-clitoromegaly is often seen in preterm newborns, where the clitoral size falls within the normal range, but the labia majora cannot completely cover labia minora, creating a misleading impression of clitoromegaly. Genital edema may also be observed in sick preterm babies or cases of ovarian hyperstimulation, which can cause pseudo-clitoromegaly.

Table 2. Clitoral Length and Width in Preterm and Term Newborns According to Gestational Age (4)
Gestational age (week)	Number of cases	Measure	Mean (mm)	+1 SD (mm)	+2 SD (mm)	+3 SD (mm)
28-30	30	Width	3.28	3.87	4.46	5.05
28-30	30	Length	5.03	5.91	6.79	7.67
30-32	57	Width	3.32	3.68	4.04	4.4
30-32	57	Length	5.03	5.52	6.01	6.5
32-34	74	Width	3.41	3.77	4.13	4.49
32-34	74	Length	5.14	5.65	6.16	6.67
34-36	104	Width	3.69	4.17	4.65	5.13
34-36	104	Length	5.53	6.12	6.71	7.3
36-38	128	Width	4.08	4.5	4.92	5.34
36-38	128	Length	5.83	6.29	6.75	7.21
>38	187	Width	4.21	4.64	5.07	5.5
>38	187	Length	6.11	6.49	6.87	7.25

GENERAL THOUGHTS ON AMBIGUOUS GENITALIA AT BIRTH

The reported incidence of ambiguous genitalia in newborns is approximately one in 4,500. It is essential that a newborn with ambiguous genitalia and their parents are promptly referred to a specialized center for evaluation. This evaluation should be carried out by a multi-disciplinary team specializing in DSD in accordance with international recommendations on diagnostic and therapeutic strategies. This team should provide holistic care and treatment guidance, collaborating closely with relevant subspecialists and peer support groups (5). Key subspecialties involved in DSD teams typically include psychology, pediatric endocrinology, urology, neonatology, gynecology, andrology, nursing, social work, genetics, and medical ethics. It's worth noting that certain aspects of DSD management remain contentious or uncertain, resulting in recent changes in clinical practice that are ongoing. Notably, genital surgery has been banned in several countries, and it is increasingly avoided in young children to protect their rights to an open future and the integrity of their bodies.

Consequently, we can anticipate a growing number of children growing up with atypical-looking genitalia, requiring a careful assessment of the psychological impact (6). Providing psychological support to affected families as a standard component of care from the outset is, therefore, of utmost importance. Issues related to gender that may arise in the context of ambiguous genitalia should be openly and thoughtfully discussed.

A comprehensive evaluation of ambiguous genitalia in a newborn is essential for understanding the underlying cause and potential consequences. On one hand, this evaluation can lead to the detection of concomitant, treatable disorders, such as adrenal insufficiency that requires cortisol replacement. Also, it is important to note that in up to 30% of children with a Difference/Disorder of Sex Development (DSD), other organ system disorders may be identified, necessitating medical care and qualifying as syndromic DSD (7, 8). On the other hand, identifying the underlying cause of ambiguous genitalia forms the basis for making an informed decision regarding sex registration at birth, if necessary and desired by the parents. Gender assignment/sex registration at birth should take into account long-term satisfaction with the sex of rearing, sexual function, and fertility potential (1, 9, 10). Ideally, it should result from a shared decision-making process involving the child's parents and the DSD network team. This decision should be based on the individual's phenotype/genotype, existing knowledge from literature, databases, and patient representative experience (11-13). This process may require time, as parents need to grasp the complexity of their child's unique biology in order to participate in this decision. As a first step, the overall health of the child should be assessed, and parents should be educated about typical sexual development before explanations regarding the underlying cause and its implications for their child's ambiguous genitalia are provided. Even if the exact details of the underlying cause remain unresolved, it is essential to communicate what is known and what remains uncertain to the parents and make plans for the family's future in the best interests of the child.

Several websites are available for patients and their families to exchange information, share coping strategies, and discuss decision-making processes that can enhance outcomes for newborns with ambiguous genitalia. Such resources can be found on websites supported by organizations like the Accord Alliance (www.accordalliance.org), DSD families (www.dsdfamilies.org), AIS DSD Support Group (www.aisdsd.org), and the CARES Foundation (www.caresfoundation.org). However, the most current information will always be available from the responsible DSD team, which will also be aware of country-specific support services.

GENETICS OF SEX DETERMINATION AND DIFFERENTIATION

Sex development is a sequential process that involves the coordinated action of numerous genes and pathways. This process culminates in the development of functional gonads, differentiated internal sex organs, external genitalia, and typical secondary sexual characteristics that manifest after puberty. Prenatal sex development can be divided into two distinct processes: sex determination and sex differentiation (Figure 3). Sex determination is the initial step that occurs shortly after conception, approximately at 6-7 weeks gestation. During this phase, the undifferentiated gonads evolve into either testes or ovaries. By contrast, sex differentiation occurs subsequently and involves hormones produced after the formation of the sex-specific gonads. These hormones play a critical role in shaping the further development of the dependent embryonic structures, leading to the formation of male and female internal and external phenotypes.

Figure 3. The genes and transcription factors required for gonadal development. The formation of the bipotential gonad requires at least seven genes including NR5A1 (Nuclear Receptor Subfamily 5 Group A Member 1), WT1 (WilmsTumor1 Transcription Factor), LHX9 (LIM Homeobox 9), EMX2 (Empty Spiracles Homeobox 2), CBX2 (Chromobox 2), PBX1 (Pre-B-Cell Leukemia Transcription Factor 1) and GATA4 (GATA Binding Protein 4). Adapted from (14).

The Male Specific Pathway

In the male specific pathway, the expression of the SRY gene in pre-Sertoli cells serves as a pivotal switch that directs the fate of the gonads toward testicular development. SRY, in conjunction with NR5A1, activates the SOX9 signaling pathway, initiating testis development (Figure 3). Other important genes for the early testis development are NR0B1(Nuclear Receptor subfamily 0 group B member 1), AMH (Anti-Mullerian Hormone) and GATA4. AMH secreted by Sertoli cells, plays a crucial role in promoting the regression of Müllerian structures and supporting the development of Leydig cells in the testes. Subsequently, androgens produced by the testes facilitate the development of male internal organs (epididymis, vas deferens, seminal vesicle) and external genitalia (penis, scrotum). The production of sex steroids relies on an intact steroidogenic pathway, with all steroid hormones originating from cholesterol. The initial steps of sex steroid biosynthesis overlap with the synthesis of mineralo-corticosteroids and gluco-corticosteroids (Figure 4). It is important to note that certain steroid disorders, often referred to as congenital adrenal hyperplasia (CAH), can affect both adrenal and gonadal steroidogenesis or result in androgen excess or deficiency that may impact fetal genital development. INSL3, produced in Leydig cells, plays a role in guiding the descent of the testes from the abdomen to the scrotal folds.

The process of masculinization of initially sex-neutral, undifferentiated external genitalia begins around 8-9 weeks of gestation when the potent androgen DHT (dihydrotestosterone) is produced by the testes. DHT is crucial for the fusion of the urethral and labioscrotal folds, elongation of the genital tubercle, regression of the urogenital sinus, and the development of the prostate (15). The enzyme 5α-reductase is responsible for converting testosterone produced by fetal Leydig cells into DHT. Complete masculinization of the external genitalia through DHT is typically achieved by week 14 of gestation (16).

Figure 4. Steroidogenesis of the adrenals and gonads. Adrenal and gonadal steroidogenesis starting from cholesterol and showing key steroids and intermediates of the classical and alternative pathways is illustrated. Cortisol and androgen biosynthesis are under control of hypothalamic-pituitary-adrenal (HPA) axis in a negative feedback loop. Androgen synthesis occurs via classical or aberrant and alternative pathways (i.e., backdoor pathway and 11oxC19 androgen pathway). Aberrant or alternative pathways are particularly active whenever cortisol with or without aldosterone biosynthesis is impaired and androgenic precursors accumulate proximal to enzymatic block. Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, CYP21A2; 21α-hydroxylase, HSD3B2; 3β-hydroxy steroid dehydrogenase 2, CYP11B1; 11β-hydroxylase, CYP11B2; Aldosterone synthase, DOC; 11-deoxycorticosterone, DHEA; Dehydroepiandrosterone, A4; Androstenedione, T; Testosterone, CYP17A1; 17α-hydroxylase/17,20-lyase, b5; CYP19A1; Cytochrome P450 aromatase, Cytochrome b5, POR; P450 oxidoreductase, 17β-HSD; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SRDA1/2; Steroid 5α -reductase 1/2, 11oxC19; 11-oxygenated 19-carbon (11oxC19) steroids; Fdx; Ferredoxin, FdR; Ferredoxin reductase, 11βOHA; 11β-hydroxyandrostenedione, 11βOHT; 11β-hydroxytestosterone, Pdione; 5α-Pregnane- 17α-ol-3,20-dione, Pdiol; 5α-Pregnane- 3α,17α-diol 20-one, AKR1C2,4; Aldo-Keto Reductase Family 1 Member C2,4, DHT; Dihydrotestosterone, HSD17B5,3,6= 17β-hydroxysteroid dehydrogenase 5,3,6. Adapted from (17).

The Female Specific Pathway

By contrast, the female-specific pathway is initiated in the absence of SRY and requires the suppression of SOX9 (Figure 3). Activation of Rspo1/Wnt4/b-catenin and Foxl2 (Forkhead Box L2) signaling pathways leads to the formation of typical ovaries. The absence of AMH allows the Müllerian structures to proliferate, resulting in the formation of the typical fallopian tube, uterus, cervix and the upper part of the vagina. In the absence of both testosterone secretion and 5α-reductase activity, the sex-neutral external genitalia develop along the female pathway. Specifically, without DHT, the labioscrotal and urethral folds give rise to the labia majora and minora, respectively. The genital tubercle develops into a clitoris, and the urogenital sinus contributes to the formation of the urethral opening and anterior portion of the vagina (16).

Any disruption in the intricate genetic and hormonal processes involved can lead to atypical sex development and result in atypical external genitalia at birth. Figure 3 provides a summary of these processes and highlights some key genes and hormones essential for typical development.

DIFFERENCES/DISORDERS OF SEX DEVELOPMENT (DSD)

The discordance between genetic, gonadal, or anatomic sex is commonly referred to as DSD. In addition to candidate gene testing, the new era of molecular diagnostic tools, including whole exome/genome sequencing has uncovered numerous novel molecular etiologies in recent years. Accurate molecular diagnosis aids in managing affected individuals and provides families with information concerning prognosis and the risk of recurrence. This information is often sourced from literature and comprehensive data registries, such as the I-DSD (18-24). For clinical purposes, DSD (including ambiguous genitalia in newborns) is typically classified based on the affected individual's karyotype. The consensus statement addressing the approach and care of DSD (originally published in 2006 and updated in 2016) suggests the following broad classification for DSD: (A) Sex Chromosome DSD, (B) 46,XY DSD, and (C) 46,XX DSD (Table 3) (1, 20).

Table 3. DSD Classification According to the Chicago Consensus.
Sex Chromosome DSD	46,XY DSD	46,XX DSD
A. 45,X0 (Turner syndrome and variants)	A. Disorders of gonadal (testicular) development Complete gonadal dysgenesis (Swyer syndrome) or partial gonadal dysgenesis (e.g., SRY,SOX9, NR5A1, WT1, DHHgene mutations etc) Testicular regression (e.g., DHX37 gene mutation) Ovo-Testicular DSD	A. Disorder of gonadal (ovarian) development Ovo-Testicular DSD Testicular DSD (SRY (+)) Gonadal dysgenesis
B. 47,XXY (Klinefelter syndrome and variants)	B. Disorders of testicular hormone production or action Impaired testosterone production LH receptor mutations CAH (3βHSD2 deficiency, 17OHD, POR, StAR, CYP11A1 deficiency) HSD17B3 deficiency Impaired testosterone action 5α-reductase deficiency Androgen insensitivity syndrome (Complete/partial) Impaired AMH production or action Persistent Mullerian Duct Syndrome (AMH and AMHR2gene mutations)	B. Androgen excess Fetal CAH (21-hydroxylase, 11β-hydroxylase, 3βHSD2 deficiency) Glucocorticoid resistance Feto-placental Aromatase deficiency POR deficiency Maternal Luteoma, hilar cell tumors, arrhenoblastoma, lipoid cell tumors, Krukenberg tumors, androgen producing adrenal tumors, External androgen exposure
C. 45,X0/46,XY (Mixed gonadal dysgenesis, ovotesticular DSD)	C. Other Syndromic Smith-Lemli-Opitz, Cloacal anomaly, Aarskog, Robinow, Meckel, Joubert, Hand-Foot-Genital, popliteal pterygium, CHARGE, VATER/VACTERL, IMAGe etc. Drugs Flutamide, ketoconazole, progestagens Endocrine disruptors Phthalate, BPA, paraben Cryptorchidism INSL3, GREAT gene mutations etc. Isolated hypospadias MAMLD1, HOXA4 gene mutations etc.	C. Other Syndromic Cloacal anomaly etc. Mullerian anomalies Mayer-Rokitanski-Küster-Hauser, MURCS, McKusick-Kaufmnann syndrome and variants, MODY5
D. 46,XX/46,XY (chimeric, ovotesticular DSD)

Abbreviations: StAR; Steroidogenic Acute Regulatory Protein defect, CYP11A1; P450 side-chain cleavage deficiency, 3βHSD2; 3β-hydroxy steroid dehydrogenase 2, CYP17A1; 17α-hydroxylase/17,20-lyase deficiency, 17OHD; 17α-hydroxylase/17,20-lyase deficiency, POR; P450 oxidoreductase, HSD17B3; 17β-hydroxysteroid oxidoreductase or 17-ketosteroid reductase; SF1/NR5A1; steroidogenic factor 1.

The 46,XY Newborn With Ambiguous Genitalia

Ambiguous genitalia in a 46XY newborn can result from abnormal formation of the early fetal testes (testicular dysgenesis), reduced production of testosterone or dihydrotestosterone (5α-reductase deficiency), or the inability to respond to androgens (androgen insensitivity syndrome, or AIS) (Table 3). Depending on the extent of androgen production defect or resistance, affected newborns may exhibit a range of external genitalia phenotype, varying from those that appear typically female to those that appear typically male but with a small phallus, hypospadias, and a bifid scrotum, with or without palpable testes. Here we focus primarily on causes that lead to ambiguous genitalia. Broadly, four groups of underlying causes of 46, XY DSD with ambiguous genitalia can be identified: (a) partial gonadal dysgenesis, (b) partial testosterone biosynthetic defects, (c) partial 5α-reductase deficiency, and (d) partial androgen insensitivity syndrome or PAIS.

PARTIAL GONADAL DYSGENESIS

In cases of partial gonadal dysgenesis, it is presumed that a gene mutation leads to a partial abnormality in the development of the early urogenital ridge or gonadal anlage. This category of DSD can also involve mutations in genes such as SRY and SOX9, which are essential for the differentiation of bipotential gonads into testes (Figure 3). Partial gonadal dysgenesis is typically associated with incomplete masculinization of the external genitalia, along with varying degrees of maintenance of Wolffian ducts and inhibition of Müllerian ducts. For a summary of known genetic variations associated with gonadal dysgenesis, please refer to Table 4.

Leydig cell aplasia or hypoplasia represents a variation of gonadal dysgenesis, characterized by inadequate Leydig cell differentiation in the male gonad, resulting in impaired androgen production. Mutations in the LH receptor gene (LHCGR) as well as polymorphisms have been associated with this condition (25). The phenotype associated with Leydig cell hypoplasia includes incomplete masculinization of the external genitalia, along with incomplete development and maintenance of the Wolffian ducts.

Table 4. Monogenic Disorders of Gonadal Development/Differentiation and Associated DSD Presentations in Humans, Both 46,XY and 46,XX
Gene defect	Locus	Mode of transmission	Clinical presentation			Reference
			DSD phenotype		Other
			46, XX	46, XY
ARX	Xp22.13	XL		Testicular dysgenesis	Lissencephaly, epilepsy, intellectual disability temperature instability	(26)
ATRX	Xq13.3	XL		Testicular dysgenesis	Dysmorphic features, intellectual disability, α-thalassemia	(27)
CBX2	17q25	AR	Ovarian dysgenesis	Testicular dysgenesis		(28)
DHH	12q13.12	AR		Testicular dysgenesis	Minifascicular neuropathy	(29)
DMRT1/DMRT2	9p24.3	AD	Ovarian dysgenesis	Testicular dysgenesis	Hypotonia, developmental delay, impaired intellectual development	(30)
DHX37	12q24.31	AD		Testicular dysgenesis, testicular regression		(31)
EMX2	10q26.11		Ovarian dysgenesis	Testicular dysgenesis	Intellectual disability, kidney agenesis	(32)
ESR2	14q23.2-q23.3	AD,?	Ovarian dysgenesis	Testicular dysgenesis	Dysmorphic features, eye abnormalities, anal atresia, rectovestibular fistula	(33)
FGFR2	10q26.13	AD		Testicular dysgenesis, ovotesticular	Craniosynostosis	(34)
GATA4	8p23.1	AD		Testicular dysgenesis	Congenital heart defects (atrial septum defects, ventricular septum defects, tetralogy of Fallot), diaphragmatic hernia	(35)
HHAT	1q32.2	AR		Testicular dysgenesis	Nivelon-Nivelon-Mabille syndrome. Dwarfism, chondrodysplasia, narrow, bell-shaped thorax, micromelia, brachydactyly, microcephaly with cerebellar vermis hypoplasia, facial anomalies, hypoplastic irides and coloboma of the optic discs	(36)
LHX9	1q31.3	AD		Testicular dysgenesis	Limb anomalies	(37)
MAMLD1	Xq28	XL		Severe hypospadias	Myotubular myopathy	(38)
MAP3K1	5q11.2	AD		Testicular dysgenesis		(39)
NR0B1 (Duplications, inversion and upstream deletion)	X21.3	XL		Testicular dysgenesis	Congenital adrenal hypoplasia, hypogonadotropic hypogonadism, cleft palate, intellectual disability	(40)
NR2F2	15q26.2	AD	Ovarian dysgenesis, ovotesticular DSD		Congenital heart disease, somatic anomalies including blepharophimosis-ptosis-epicanthus inversus syndrome	(41)
NR5A1	9q33	AD	Ovarian dysgenesis, ovotesticular DSD	Testicular dysgenesis	Adrenocortical insufficiency, spermatogenic failure, primary ovarian insufficiency, asplenia, polysplenia	(42)
PPP2R3C	14q13.2	AR		Testicular dysgenesis	Dysmorphic facies, retinal dystrophy, and myopathy.	(43)
RSPO1	1p34.3	AR	Testicular/ ovotesticular DSD		Palmoplantar hyperkeratosis	(44)
SART3	12q23.3	AR		Testicular dysgenesis	Intellectual disability, global developmental delay and a subset of brain anomalies	(45)
SOX3 (Upstream deletion and Duplication)	Xq27.1	XL	Testicular/ovotesticular DSD		Intellectual disability, hypopituitarism	(46)
SOX8 (SNV, upstream duplication, inversion)	16p13.3	AD		Testicular dysgenesis	Variants may contribute to male and female infertility, primary ovarian insufficiency.	(47)
SOX9 (SNV, upstream duplication, inversion)	17q24.3	AD	Testicular/ovotesticular DSD	Testicular dysgenesis	Campomelic dysplasia, Cooks syndrome, Pierre Robin sequence	(48)
SOX10 (Duplication)	22q13.1	AD	Testicular/ovotesticular DSD		Peripheral and central demyelination, Waardenburg syndrome type IV, and Hirschsprung disease	(49)
SRY (SNV, deletion, Translocation)	Yp11.3	XL, YL	Testicular/ovotesticular DSD	Testicular dysgenesis		(50, 51)
TSPYL1	6q22.1	AR		Testicular dysgenesis	Sudden infant death syndrome	(52)
WT1	11p13	AD	Testicular/ovotesticular DSD	Testicular dysgenesis	Denys-Drash syndrome; Frasier syndrome; WAGR syndrome; Meacham syndrome, Nephrotic syndrome; Wilms tumor	(53, 54)

Abbreviations: AD; autosomal dominant, AR; autosomal recessive, XL; X-linked, YL; Y-linked

DEFICIENCY OF TESTOSTERONE BIOSYNTHESIS

The inability to produce testosterone stems from defects in the activity of any of the enzymes required for testosterone biosynthesis from cholesterol (Figure 4able 5). Identified defects encompass genetic variants in Steroidogenic Acute Regulatory Protein (StAR), P450 side-chain cleavage (CYP11A1), 3β-hydroxy steroid dehydrogenase 2 (HSD3B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and P450 oxidoreductase (POR) (59). In addition, apparent lyase deficiency due to variants in cytochrome b5 (CYB5) and variants in AKR1C2/4 have been very rarely described (60-62). Moreover, 17β-hydroxysteroid dehydrogenase deficiency (also known as 17β–hydroxysteroid oxidoreductase or 17-ketosteroid reductase, HSD17B3) is another cause of 46,XY DSD (63). Furthermore, variants in steroidogenic factor 1 (SF1/NR5A1), which regulates the transcription of several genes involved in steroidogenesis, may lead to ambiguous genitalia. Notably, variants in HSD3B2, POR, and NR5A1 can result in ambiguous genitalia in both 46,XY and 46,XX newborns (64-66).

Similar to partial gonadal dysgenesis, a partial testosterone biosynthesis defect leads to ambiguous external genitalia and variable degrees of Wolffian duct development. However, unlike partial gonadal dysgenesis, Müllerian ducts are not retained in 46,XY newborns with partial testosterone biosynthesis defects due to intact Sertoli cell function and normal AMH production.

Table 5. Adrenal and/or Gonadal Causes of Impaired Sex Steroid Biosynthesis Associated with Ambiguous Genitalia in 46,XX and 46,XY Individuals
Disorder	Gene/ OMIM	Adrenal Insufficiency	46,XY Gonadal Phenotype (Testosterone Deficiency)	46,XX Gonadal Phenotype (E2 Deficiency)	Fertility	Other Features
Lipoid congenital adrenal hyperplasia (LCAH)	StAR 201710	YES	Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal or NK	Classic: primary or secondary ovarian insufficiency (POI) Non-classic: NK or normal	Classic:Absent in 46,XY; variable in 46,XX
P450 side chain cleavage syndrome (CAH)	CYP11A1 118485	YES	Classic form: 46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal	Classic: primary or secondary ovarian insufficiency (POI) Non-classic: NK or normal	Reported in 46,XX
3β-hydroxysteroid dehydrogenase II deficiency (CAH)	HSD3B2 201810	YES	46,XY DSD, gonadal insufficiency; ambiguous genitalia Non-classic form: normal, but premature adrenarche	46,XX DSD with atypical genital development; gonadal insufficiency; ambiguous genitalia Non-classic form: normal, but premature adrenarche	Absent in 46,XY; reported in 46,XX
21-hydroxylase deficiency (CAH)	CYP21A2 201910	YES	Classic form: normal Non-classic form: normal	46,XX DSD with atypical genital development; Non-classic form: premature adrenarche, virilization, PCO	Normal in both 46,XX and 46,XY, if treated	Cave: Testicular adrenal rest tumor (m>>f) CAH-X (when combined with Ehlers-Danlos syndrome with contiguous gene variants)
11-hydroxylase deficiency (CAH)	CYP11B1 202010	YES	Classic form: normal Non-classic form: normal	46,XX DSD with atypical genital development; Non-classic form: premature adrenarche, virilization, PCO	Normal in both 46,XX and 46,XY, if treated	Hypertension
Combined 17-hydroxylase, 17,20 lyase deficiency (CAH)	CYP17A1 202110	Rare	46,XY DSD, gonadal insufficiency; atypical genital development	Lack of pubertal development, POI	Possible in 46,XX with assisted fertility measures	Hypertension and hypokalemic alkalosis (not seen with isolated lyase deficiency)
P450 oxidoreductase deficiency (CAH)	POR 124015 201750	Variable	Mild to severe 46,XY DSD, gonadal insufficiency; atypical genital development	46,XX DSD with atypical genital development or premature adrenarche, virilisation, POI, PCO	Reported	Maternal virilization during pregnancy; Antley-Bixler skeletal malformation syndrome; changes in drug metabolism
Cytochrome b5 deficiency	CYB5A 613218	NO	46,XY DSD; atypical genital development	NK	NK	Methemoglobinemia
17β-hydroxysteroid dehydrogenase III deficiency / 17-ketosteroid reductase deficiency	HSD17B3 264300	NO	46,XY DSD; atypical genital development; progressive virilization and gynecomastia at puberty	Normal	Decreased or absent in 46,XY
5α-reductase II deficiency	SRD5A2 607306	NO	46,XY DSD; atypical genital development; progressive virilisation and gynecomastia at puberty	Normal	Impaired in 46,XY
3α-hydroxysteroid dehydrogenase deficiency	AKR1C2/4 600450 600451	NO	46,XY DSD; gonadal insufficiency; atypical genital development	Normal	NK
Aromatase deficiency	CYP19A1 107910	NO	Normal	46,XX DSD with variable degree of virilisation at birth (ambiguous genitalia), gonadal insufficiency, POI	Impaired in 46,XX	Overgrowth and metabolic anomalies in males
Steroidogenic factor 1	NR5A1/ SF1 184757	Rare	Mild to severe 46,XY DSD; gonadal insufficiency – very variable; ambiguous genitalia	POI or normal; rarely 46,XX DSD with atypical genital development	Mostly impaired in 46,XY; variable in 46,XX

Abbreviations: NK; not known, POI; primary ovarian insufficiency, PCO; polycystic ovary.

5-ALPHA-REDUCTASE-2 DEFICIENCY (SRD5A2 VARIANTS)

Deficiency of the 5α-reductase enzyme arises from variants in the steroid 5α-reductase type 2 (SRD5A2) gene, which can show single point mutations to complete gene deletions. Newborns affected by this condition possess functioning Leydig and Sertoli cells, but due to their inability to convert testosterone to DHT, they may exhibit varying degrees of under-masculinized external genitalia, including genital ambiguity at birth in some cases (16, 67-69). The Müllerian ducts in affected individuals regress as expected, due to normal Sertoli cell function and AMH production. During puberty, significant virilization becomes possible in affected individuals, and the testes are capable of supporting spermatogenesis, as DHT is not required for germ cell maturation. Therefore, fertility is attainable in less severely affected individuals (70-72) or with the use of intrauterine insemination. Recent evidence also suggests that SRD5A2 activity may be even influenced by genetic polymorphisms of SRD5A2 (50).

GENETIC DEFECTS OF ANDROGEN ACTION

Complete or partial androgen insensitivity syndromes (CAIS/PAIS) are caused by genetic mutations affecting androgen receptor (AR) function (73, 74), and serum testosterone levels are typically elevated. Internationally, incidence of AIS is reported in 1 in 20,400 live born 46,XY infants, with CAIS occurring at a higher rate than PAIS (75).

The human androgen receptor (AR) is encoded by a single gene (AR) composed of 8 exons located in the q11-12 region of the X chromosome. The AR gene mutation database includes hundreds of AR variants that lead in varying degrees of atypical sex differentiation of 46,XY fetuses, also classified as AIS type I (76-78). A small number of complete AR gene deletions have been reported, as well as deletions that start at exons 2, 3 or 4 and extend to the terminus of the gene. In addition, a limited number of mutations resulting from premature terminations, base deletions and terminations have been identified. However, the most common type of AR gene mutation results from base substitutions (79). When mutations result in partial inactivation of AR activity (PAIS), the phenotypic variability in under-masculinization can be substantial and depends on the residual activity of the AR. In some cases, this can lead to ambiguous genitalia in newborns with a 46,XY chromosomal sex.

Unlike in CAIS, the underlying genetic defect in PAIS is only found in the AR gene in about 40% of cases (80). However, the clinical and biochemical phenotype is similar, and in vitro functional tests demonstrate androgen resistance in all of them. Thus, individuals with AIS without AR mutations are classified as AIS type II (73). In cases of AIS type II without AR variants, largely unidentified regulators or cofactors of the AR are responsible for the impaired AR signaling, as revealed by an AR-dependent bioassay using genital skin fibroblasts and the targeted apolipoprotein D as a biomarker (81). Given that AR activity can be regulated at various levels, the potential mechanisms of AIS type II are diverse.Recently, altered DNA methylation of the AR promoter has been observed in some individuals with PAIS (82). Additionally, mutations in the Disheveled associated activator of morphogenesis 2 gene (DAAM2) that impair nuclear actin assembly at AR have been associated with AIS type II (83).

Overall, individuals with PAIS experience variable degrees of end-organ unresponsiveness to androgens, resulting in varying degrees of Wolffian duct development and external genital ambiguity. Within families with the same AR mutation, phenotypic variability can occur due to variable degrees of insensitivity to androgens (84, 85). Consequently, solely relying on genetic information, it is challenging to predict whether a newborn with PAIS will identify as male, intersex, or female in later life and respond to future testosterone therapy. Similarly, genetic counselling is challenging.

The 46,XX Newborn With Ambiguous Genitalia

Ambiguous genitalia in a chromosomal 46,XX fetus invariably results from androgen excess during fetal development, and the degree of virilization (Figure 2) often provides clues about the timing and severity of the underlying disorder.

Similar to a 46,XY fetus, a 46,XX fetus may experience partial gonadal dysgenesis or develop testicular or ovo-testicular DSD due to pathogenic variants in genes involved in early sex determination (Table 3 and 4). Ovo-testicular DSD occurs when both ovarian and testicular tissue develop in the same individual (1). Most newborns with ovo-testicular DSD possess 46,XX chromosomes and present with ambiguous genitalia, although some affected individuals have 46,XY chromosomes or 46,XX/46,XY mosaicism. Just like in 46,XY DSD, the degree of testicular development determines the extent of masculinization/virilization of the external genitalia, Wolffian duct development, and Müllerian duct regression in affected newborns (79). A summary of known genetic variations is given in Table 4.

In the majority of cases, ambiguous genitalia in a 46,XX fetus with typical ovarian organogenesis can be attributed to excessive androgen production/exposure early in utero, originating either from the fetus itself (Table 3 and Table 5) or from the mother or environment.

ABNORMAL FETAL ANDROGEN PRODUCTION BY CONGENITAL ADRENAL HYPERPLASIA (CAH)

The term congenital adrenal hyperplasia (CAH) encompasses several adrenal disorders, each linked to a mutation in one of the enzymes necessary for the biosynthesis of cortisol from cholesterol (86) (Figure 4 and Table 5). These abnormalities lead to increased ACTH secretion by the pituitary gland, which, in turn, results in the increased secretion of cortisol precursors, including adrenal androgens.

Deficiency of steroid acute regulatory protein (StAR) causes congenital lipoid adrenal hyperplasia, characterized by salt loss and a lack of cortisol, androgen, and estrogen secretion. Genetic 46,XX infants affected by StAR deficiency exhibit typical female external genitalia, while genetic 46,XY are born with ambiguous genitalia or may experience sex reversal in cases of severe loss-of-function variants. A similar phenotype is also observed with variants in CYP11A1.

Deficiency in 17α-hydroxylase/17,20-lyase leads to hypertension due to the hypersecretion of corticosterone and impaired androgen secretion. Similar to congenital lipoid adrenal hyperplasia, 46,XX fetus affected by 17α-hydroxylase/17, 20-lyase deficiency (CYP17A1) develop typical female external genitalia, while 46,XY fetus present with ambiguity.

Deficiency of 3β-hydroxysteroid dehydrogenase (HSD3B2) results in salt loss and impaired androgen synthesis. Affected 46,XX females may or may not exhibit minimal genital masculinization, whereas all genetic 46,XY males are variably under-masculinized (87).

CAH due to 11β-hydroxylase deficiency (CYP11B1) and 21-hydroxylase deficiency (CYP21A2) results in the most significant masculinization/virilization of external genitalia in 46,XX fetus compared to all other types of CAH. Additionally, 11β-hydroxylase deficiency can lead to hypertension in individuals of either sex in later life (88, 89).

21-hydroxylase deficiency CAH, caused by variants in CYP21A2, represents the most common form (accounting for more than 90% of cases) of CAH and is the most frequent genetic cause of DSD with ambiguous genitalia at birth in 46,XX newborns (59, 63). Cortisol deficiency necessitates replacement therapy in all classic forms of CAH. In the milder (simple-virilizing) presentation of classical 21-hydroxylase deficiency, salt loss is typically not an issue, whereas in the more severe (salt-wasting) form of classical 21-hydroxylase deficiency, salt-loss occurs and may pose a risk to the newborn shortly after birth if not treated with both mineralocorticoids and glucocorticoids (86, 90, 91).

The pathological mechanisms of 21-hydroxylase deficiency leading to 46,XX virilization in utero have been well described (92). It involves androgen excess due to lack of 21-hydroxylase activity during the critical time window around 6-12 weeks of gestation when the external genitalia are formed, and adrenal androgen production should be redirected to cortisol production to protect the female external genital anlage. In 46,XX CAH (Table 5), the resulting adrenal androgen excess exceeds the androgen metabolizing capacities of the fetal-placental unit, and alternative pathways of androgen production (Figure 4) are also activated, contributing to the overall androgen excess. Figure 5 illustrates in a scheme how the fetal adrenals, liver and placenta cooperate under normal conditions to metabolize fetal adrenal androgens into estrogens.

Figure 5. Steroid metabolic pathways in the fetal-placental-maternal unit, also known as the fetal-placental unit. The main function of the FZ of the fetal adrenal is the production of DHEA-S from cholesterol, which is transported to the placenta, desulfated to DHEA, and sequentially metabolized by placental 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), 17β-hydroxysteroid dehydrogenases (17βHSD) and cytochrome P450 aromatase (CYP19A1), to androstenedione (A4), testosterone (T) and estradiol (E2), respectively. E2 is then transported from the placenta into the maternal circulation. A4 is additionally also a substrate for placental CYP19A1, producing estrone (E1). Fetal adrenal DHEA-S is also metabolized by the fetal liver to 16α-hydroxy-DHEA-S by cytochrome P450 family 3 subfamily A member 7 (CYP3A7), which is metabolized to estriol (E3), the estrogen marker of pregnancy. Abbreviations: PREG, pregnenolone; 17OHPREG, 17α-hydroxypregnenolone; PROG, progesterone; DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone-sulfate; CYP11A1, cytochrome P450 cholesterol side chain cleavage; StAR, steroidogenic acute regulatory protein; CYP17A1, cytochrome P450 17α-hydroxylase/17,20-lyase; SULT2A1, sulfotransferase; CYP3A7, cytochrome P450 family 3 subfamily A member 7; CYP19A1, cytochrome P450 aromatase; HSD17B, 17β-hydroxysteroid dehydrogenase; HSD3B1, 3β-hydroxysteroid dehydrogenase type 1; HSD11B2, 11β-hydroxysteroid dehydrogenase type 2; STS, sulfatase.

FETAL AND PLACENTAL AROMATASE DEFICIENCY

During fetal development, the adrenals produce substantial amounts of DHEA/S. These are either converted to 16-hydroxy-DHEA/S in the fetal liver and then transported or directly transferred to the placenta, where they are converted into estrogens (Figure 5). In case of an aromatase enzyme deficiency in the fetus (caused by variants in the CYP19A1 gene), androgen precursors accumulate and lead to virilization of 46,XX fetuses, typically leading to ambiguous genitalia at birth (93). Androgen excess due to fetal aromatase deficiency may also induce maternal virilization during pregnancy, as indicated by a deepening of the voice, oily skin and hair, and excessive hair growth (hirsutism).

EXCESS MATERNAL ANDROGEN PRODUCTION

Excessive androgen production can negatively impact fertility due to anovulation, therefore cases of maternal androgen production during pregnancy are exceedingly rare. Nonetheless, androgens originating from the mother can travers the placenta and lead to the masculinization of a female fetus. These maternal androgens usually originate from the ovaries or adrenal glands. Androgen-producing ovarian tumors include hilar cell tumors, arrhenoblastomas/androblastomas, lipoid cell tumors, and Krukenberg tumors. In contrast, pregnancy luteomas, rare non-neoplastic ovarian lesions occurring during pregnancy, are believed to be caused by hormonal effects related to pregnancy. Although exceptionally rare, androgen-secreting tumors of the adrenals can also occur during pregnancy.

DRUGS ADMINISTERED TO THE MOTHER DURING GESTATION

In 1958, Wilkins et al. reported that certain synthetic progestins administered to pregnant women, such as 17α-ethinyl-19-nortestosterone, can masculinize the external genitalia of female fetuses (94). Similarly, diethylstilbesterol (DHE), a nonsteroidal synthetic estrogen, has been linked with urogenital anomalies in both 46,XX and 46,XY fetuses (95, 96). More recently, numerous studies have indicated that fetal exposure to various endocrine disrupting chemicals (EDCs) such as pesticides, fungicides, herbicides, and plasticizers may adversely affect genital development (97-99).

Syndromes Associated with Ambiguous Genitalia

As previously mentioned in sections of 46,XX and 46,XY DSD, ambiguous genitalia can be associated with syndromes characterized by multiple congenital malformations, as summarized in Table 4. These associations arise because many of the transcription factors involved in sex development and differentiation also have roles in other aspects of development (Table 4).

For example, mutations of the WT-1 gene (11p3) can lead to several syndromes, including WAGR, Denys-Drash or Frasier syndromes (70-72). Mutations in SOX-9 (17q24-25) can result in campomelic dysplasia (100).

Mutations in DMRT1/DMRT2 (9p24.3), EMX2 (10q25.3q26.13), ATRX (Xq13.3) and WNT-4 (1p35) are often associated with developmental delay (101, 102). SF-1 (9q33) and DAX-1 (Xp21.3) play roles in the early formation of the adrenals, anterior pituitary, and parts of the hypothalamus.

Consequently, mutations can lead to abnormalities of these organs, in addition to those of the urogenital system. Abnormal development of the lower abdominal wall with pubic diastasis can result in urogenital anomalies observed in the VA(C)TER(L) and CHARGE syndromes, as well as bladder and cloacal exstrophy (103).

These examples underscore the complexities of the anomalies that may be associated with ambiguous genitalia in newborns and emphasize the importance of conducting a thorough physical examination and involving an experienced DSD team when faced with a child presenting ambiguous genitalia.

WORKUP OF NEWBORNS WITH AMBIGUOUS GENITALIA

The etiology of genital ambiguity in newborns is diverse and can have significant implications for management. Therefore, obtaining an accurate diagnosis through a medical workup is crucial. Some investigations are urgent, such as ruling out or confirming and treating potentially life-threatening adrenal insufficiency with mineralocorticoid and glucocorticoid replacement therapy in children with underlying CAH. Additionally, associated organ anomalies may require immediate attention to ensure the child’s survival, such as cardiac, pulmonary, or kidney malfunctions.

When the newborn is shown to be capable of excreting urine and stool without problems, and adrenal and other organ functions are found to be intact, investigations to understand genital ambiguity become primarily a psychosocial “emergency” for the family and caregivers. As a result, psychological support and holistic care by a DSD team as early as possible are essential to guide the parents and make decisions in the child’s best long-term interest. Obtaining an accurate diagnosis of the underlying cause of genital ambiguity early on is necessary for decision-making processes regarded to sex registration, hormonal and surgical treatments, and outcomes such as potential fertility and risk of gonadal malignancy. Therefore, it is recommended practice that a thorough workup should be performed for every child born with ambiguous genitalia, and guidelines for such assessments are available (22, 104-107).

Overall Assessment of the Child with Ambiguous Genitalia

Figure 6 provides an overview of the initial workup for a newborn with ambiguous genitalia. Several aspects are considered, including pregnancy and family history, as well as postnatal adaptation (such as feeding, weight gain, alertness, excretion). In some cases, suspicion of genital ambiguity may arise during pregnancy through ultrasound surveillance. In such cases, first contact and information through a DSD team member (e.g., neonatologist, geneticist, pediatric endocrinologist) together with the DSD psychologist is recommended to guide the parents through pregnancy and alert the future care team early. Prenatal workup of ambiguous genitalia is generally not necessary if the fetus is otherwise developing normally without other organ anomalies that require attention. It is important to note that a fetus with CAH does not suffer from mineralocorticoid and cortisol deficiency in utero, as these hormones become essential only after birth (Figure 4). Genetic workup of a child with ambiguous genitalia during the prenatal period may only be recommended when there is a previous index case with a diagnosis, or when parental genetic data inform an abnormality, and when genetic counseling of the parents has led to a personalized action plan.

When taking the medical history of a newborn with ambiguous genitalia, asking specific questions can help guide the diagnostic workup and management. Figure 6 provides a list of important questions to consider. For example, androgenic drugs used in pregnancy may explain the virilization of a 46, XX newborns. Maternal virilization during pregnancy, for example, voice change and hirsutism may be important for the diagnoses of maternal virilizing tumors, P450 oxidoreductase (POR) deficiency, or placental aromatase deficiency. Questions about parental consanguinity are important, because many of the causes of ambiguous genitalia are recessively inherited. History of previously unexplained neonatal deaths, ambiguous genitalia, infertility or genital surgery in the family is important for identifying potential genetic etiologies.

In addition to the medical history, every newborn with ambiguous genitalia should undergo a comprehensive physical examination, with a special focus on signs of adrenal insufficiency (dehydration, lethargy, poor feeding) and any dysmorphic features (Figure 6). This examination may guide the diagnostic process.

Figure 6. Algorithm for initial workup of the child with ambiguous genitalia at birth.
Abbreviations: SDSST; Standard dose synthetic ACTH (synacthen) stimulation test, HCG; Human chorionic gonadotropin, WES; Whole exome sequencing, WGS; Whole genome sequencing, EMS; External masculinization score, EGS; External genital score, QF-PCR; quantitative fluorescence polymerase chain reaction, SRY FISH; SRY fluorescent in situ hybridization.

Physical Examination of the External Genital Area

Systematic assessment of the external genital area in newborns with ambiguous genitalia is crucial, and it should be carefully documented. Over the years, different scales and scoring systems have been used to evaluate the external genitalia. These assessments help provide a standardized way to document the physical characteristics, which is essential for comparing data over time and between different healthcare providers and patients. The use of these scales and scoring systems aids in follow-up and tracking of changes in the external genitalia.

Historically, the Prader or Quigley scales were commonly used for this purpose (108, 109) (Figure 7). Later, the external masculinization score (EMS) was introduced as a more comprehensive assessment tool (104) (Table 6). Nowadays, the external genital score (EGS) is recommended for evaluating and documenting the external genitalia (110) (Table 7). These tools provide a structured way to assess and record the physical findings, making it easier to monitor and communicate about the patient's condition.

The careful examination of the external genitalia in newborns with ambiguous genitalia provides valuable non-invasive, indirect information about prenatal exposure and response to DHT, and about the timing of this exposure. For instance, androgen exposure must have occurred in the first trimester to cause labial fusion.

During the genital exam, it’s important to measure the stretched phallic length, assess the quality of the corpora (erectile tissues), inspect of the labia, labio-scrotal folds, or scrotum, and document the position of the urethral opening (and vaginal opening, if applicable). Furthermore, the presence and location of palpable gonads should also be recorded. Any asymmetry in the gonads and external genitalia should be noted. Asymmetry in internal and/or external genital structures may suggest mixed gonadal dysgenesis or ovo-testicular DSD. All these observations can be documented using the external genital score (EGS), which provides a standardized way to assess and record the physical characteristics of the external genitalia (Table 7). In addition, the anogenital distance (AGD), the distance between the anus and the base of the genitalia, can be helpful in determining the extent of androgen exposure during fetal development, as a higher ratio is generally consistent with increased androgen exposure (111) (Table 8). In older infants, the anogenital index (AGI), which is the body weight standardized index of AGD [AGI/weight (mm/kg)], may be used for assessments to account for changes of the external genitalia through growth and postnatal development (111).

Overall, a thorough examination and documentation of the external genitalia provide important clinical information that can guide the diagnosis and management of newborns with ambiguous genitalia.

Figure 7. Prader and Quigley Scales for visual grading of external genital virilization. In Stage 3 a single urogenital orifice can be observed.

Table 6. External Masculinization Score (EMS)
External Masculinization Score (EMS).	Scrotal fusion	Micropenis	Urethral meatus	Right gonad	Left gonad
3	Yes	No	Normal
2			Distal
1.5				Labioscrotal	Labioscrotal
1			Mid	Inguinal	Inguinal
0.5				Abdominal	Abdominal
0	No	Yes	Proximal	Absent	Absent

An EMS score <11 out of 12 needs further clinical investigation for DSD in 46,XY.

Table 7. External Genital Score (EGS)
External genital score (EGS)	Labio-scrotal fusion	Genital tubercle size (mm)	Urethral meatus	Right gonad	Left gonad
3	Fused	≥31	Typical male
2.5		26-30	Coronal glandular
2			Penile
1.5	Posterior fusion	21-25	Peno-scrotal	Labio-scrotal	Labio-scrotal
1		10-20	Scrotal	Inguino-scrotal	Inguino-scrotal
0.5			Perineal	Inguinal	Inguinal
0	Unfused	≤ 10	Typical female	Impalpable	Impalpable

Median EGS (10th to 90th centile) in males < 28 weeks gestation is 10 (8.6-11.5); in males 28-32 weeks 11.5 (9.2-12); in males 33-36 weeks 11.5 (10.5-12) and in full-term males 12 (10.5-12). In all female babies, EGS is 0 (0-0).

Table 8. Reference Ranges for Anogenital Distance and Anogenital Index in Term Neonates
	5th	10th	25th	50th	75th	90th	95th
Males
AGD (mm)	18.47	19.44	21.00	22.68	25.20	27.80	29.08
AGI (mm/kg)	5.59	5.83	6.30	6.93	7.62	8.22	8.57
Females
AGD (mm)	9.40	9.85	10.55	11.65	12.60	13.45	14.10
AGI (mm/kg)	3.04	3.15	3.38	3.59	3.80	4.00	4.19

Anogenital index (AGI): the body weight standardized index of AGD [AGI=AGD/weight (mm/kg)] and found that using AGI as parameter has a better correlation with age.

Basic Auxiliary Investigations in the First Days

In the first few days of life, basic investigations are essential to evaluate adrenal and gonadal development and function in otherwise healthy newborns with ambiguous genitalia. Some key aspects to address include serum electrolytes and glucose levels. They should be monitored daily as cortisol deficiency can manifest as hypoglycemia in newborns affected by CAH. Body weight and feeding characteristics should also be monitored as excessive weight loss may indicate dehydration and electrolyte disturbances.

KARYOTYPE

Besides careful clinical evaluation, first-line investigation of an individual with DSD involves confirming the chromosomal sex using quantitative fluorescence polymerase chain reaction (QF-PCR) and karyotype (22). QF-PCR detects a series of markers on the sex chromosomes and has a turn-around time of about 1–2 days. This technique has largely replaced fluorescence in situ hybridization (FISH) methods. The analysis of karyotype is a cytogenetic technique (involving G-banding) and is crucial for the initial classification of a newborn with ambiguous genitalia (Table 3). Some centers have replaced karyotyping by array-comparative genomic hybridization (aCGH) or SNP array, with faster turn-around times of 5–10 days. But these techniques will not detect structural chromosomal rearrangements and may be less effective at detecting sex chromosome mosaicism.

HORMONE STUDIES

Detailed hormone studies may be indicated including serum gonadotropins (LH, FSH), androgens and androgen precursors (17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, testosterone, dihydrotestosterone), adrenal steroids and hormones (cortisol, aldosterone, and their precursors; ACTH, renin), and anti-Müllerian hormone (AMH) (1, 24).

Care must be taken when interpreting the results in premature babies, in whom these studies may need to be repeated at a later age and special normative value may apply. Even in term babies the timepoint and the clinical context (mode of delivery, drug effects, actual stress level etc.) need to be considered when evaluating hormonal data immediately after birth. Hormonal data change massively during delivery (e.g., surge) and within days thereafter. Additionally, it's essential to be aware that normative values for hormone levels can vary depending on the laboratory and the methods used for testing. The interpretation of hormonal data should be done by experienced healthcare providers and, if necessary, in consultation with a specialist in DSD.

IMAGING STUDIES

Ultrasound imaging of the abdominal-pelvic organs is a valuable diagnostic tool in the evaluation of newborns with ambiguous genitalia. It provides important insights into the internal reproductive and urinary structures, helping to determine the presence and characteristics of gonads, Müllerian structures (e.g., the uterus), and any associated abnormalities of the urinary tract. It can be performed at the first visit of a child with ambiguous genitalia. Of course, this investigation is not easy and may only be performed by an experienced ultra-sonographer to reveal meaningful results. Ultrasound can also detect abnormalities of the urinary tract (kidney, ureters, bladder) that sometimes occur in conjunction with genital ambiguity. Interestingly, data show that a child with a uterus and a female internal system that presents with ambiguous genitalia without gonads in the genital folds and has a 46,XX karyotype, will have in 80% of cases a diagnosis of a 46,XX CAH (most likely 21-hydroxylase deficiency). The information needed to come to this conclusion in a newborn with ambiguous genitalia can be available within 48 hours in institutions with an experienced DSD team.

In a second step, usually after months or years, additional imaging studies with MRI can be helpful in identifying both the type and extent of internal sex organ development in more details. An MRI may reveal further details on Müllerian structures (uterus remanent, fallopian tubes, upper portion of the vagina) than an ultrasound and can also help for localizing abdominal gonads. MRI may also be used for surveillance of the gonad at risk for cancer.

A genitogram had been used in the past (often early) to visualize the urinary tract, and to determine its position in relation to the vagina or vagino-utricular pouch but is no longer performed in most institutions.

However, in follow-up, cystoscopy and vaginoscopy may be used. These investigations are invasive procedures requiring general anesthesia, and therefore need to have a clear indication. Often, they are performed after complications such as urinary tract infections, to better understand the anatomy and its consequences, and in preparation for a surgical procedure (112).

Overall, the choice of imaging studies and procedures should be guided by the individual's clinical presentation, diagnosis, and the specific clinical questions that need to be addressed. In many cases, a multi-disciplinary DSD team collaborates to determine the most appropriate imaging approach to provide a comprehensive evaluation and guide treatment decisions.

GENETIC STUDIES

The rapid advancement of genetic technologies, particularly massive parallel sequencing methods like DSD panels, whole exome sequencing (WES), and whole genome sequencing (WGS), has significantly expanded our understanding of the genes involved in sex development. As a result, genetic workup has become an integral part of the recommended routine evaluation for individuals with DSD and thus ambiguous genitalia at birth (8, 22, 24, 113, 114).

While making a precise genetic diagnosis is still a challenge in some cases, the use of advanced genetic techniques has improved the diagnostic yield. Currently, a clear molecular-level diagnosis can be established in approximately half of individuals with 46,XY DSD who undergo genetic workup. However, as genetic technologies continue to evolve, and our understanding of sex development genes expands, it is likely that more cases, including those involving individuals with ambiguous genitalia at birth, will receive accurate and valid diagnoses. Therefore, re-review of genetically unsolved cases by a DSD team and genetic re-analysis by DSD-specialized teams is recommended to achieve a final diagnosis in as many as possible.

In summary, the integration of genetic workup, in combination with ongoing advancements in technology and genetic knowledge, is expected to enhance our ability to provide individuals with DSD, including those with ambiguous genitalia at birth, with more precise and comprehensive diagnoses in the future. This progress will lead to better-informed clinical decisions and management strategies for newborns with ambiguous genitalia.

DIFFERENTIAL DIAGNOSIS

Generally, the result from the karyotype plays a major role in the differential diagnosis and workup of DSD and thus ambiguous genitalia (Table 3). In most cases, the chromosomes in newborns with ambiguous genitalia will be either 46,XX or 46,XY. In rare instances, chromosomal aberration such as 45,X/46,XY or 46,XX/46,XY mosaicism may be the underlying cause explaining the phenotype. Additionally, measurements of specific hormones, such as serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex steroids, AMH, inhibin B at birth and at follow-up, in the so-called mini-puberty of infancy period, can provide valuable information for the differential diagnosis of DSD and ambiguous genitalia.

In the following we provide two flowcharts and give some general information for the step-by-step workup of newborns with ambiguous genitalia and either 46,XX or 46,XY chromosomes.

46, XX DSD

A 46, XX karyotype in a newborn with ambiguous genitalia indicates that the child is a genetic female who was exposed to excessive amounts of androgens during fetal life. A flowchart for differential diagnosis is given in Figure 8.

Marked elevation of plasma 17-hydroxypregnenolone, 17-hydroxyprogesterone, 21-deoxycortisol and androstenedione, along with male-typical levels of testosterone, are characteristic of 21-hydroxylase deficiency (CYP21A2). High values of corticosterone and 11-deoxycortisol, along with elevated androgens, indicate 11β-hydroxylase deficiency (biallelic mutations in CYP11B1). In 3beta-HSD deficiency (biallelic mutations in HSD3B2), calculation of ratios of delta-5 (pregnenolone, 17OH-pregnenolone, DHEA) over delta-4 (progesterone, 17OH-progesterone, androstenedione) steroid metabolites (see Figure 4) will point towards the genetic defect.

Overall, comprehensive steroid profiling from blood or urine using chromatographic, mass spectrometric methods allow the diagnosis of most steroidogenic defects causing androgen excess CAH in 46,XX newborns (115-117) (see also Table 5). In addition, they have led to the description of novel alternate pathways to produce active androgens in the intermediate metabolism through the backdoor or 11-oxygenated pathways, especially in disorders such as 21-hydroxylase or POR deficiencies, thereby enhancing our understanding and diagnostic possibilities (115, 117, 118)(Figure 4). However, these methods are not (yet) widely available, and the interpretation of these data is not trivial. In addition, instruments and methods vary from lab to lab. Thus, method validation is tedious and comparison between labs difficult so far (119-121).

When excess maternal androgen production is the underlying cause for masculinization of a female fetus, the source of these steroids is eliminated postnatally. Thus, the various steroids studied in affected newborns will be in the female-typical range despite masculinization of the external genitalia. Then, detailed examination of the mother may provide the explanation.

Likewise, in cases of mild POR deficiency or aromatase deficiency, in which the feto-placental unit was not able to metabolize the fetal androgens and led to ambiguous genitalia, the problem will not (always) be visible in the newborn’s steroid metabolome, and the diagnosis may only be made by genetic testing. If not, only in later life, when aromatase activity is needed for estrogen production in puberty, or when ovarian cysts hint an underlying problem, the diagnosis may be made (Figure 8).

In individuals with ovo-testicular DSD and 46,XX chromosomes, masculinization arises from androgens secreted by the testicular portion of the differentiated gonads. Androgen production is similar to that produced by testes in unaffected males except that the amount is usually smaller. The degree of masculinization of the genitalia is thus linked to the amount of functioning testicular tissue, which can be determined by a serum AMH measurement (Table 9) (122).

Very low values of AMH and female-typical values for LH/FSH (Tables 9 and 10) are expected in 46,XX newborns with masculinized/virilized genitalia that is attributed to CAH or excess maternal androgen production during gestation (122, 123). AMH is higher in newborns with ovo-testicular DSD, due to Sertoli cell development in the testicular portion of their gonads where AMH is mainly produced.

On occasion, translocation of the pseudo-autosomal part of the Y chromosome along with a mutated SRY gene to an X chromosome occurs. The result is partial masculinization of the genitalia in a 46,XX newborn. With maturity, the phenotype of affected individuals closely resembles that of boys and men with Klinefelter syndrome.

Figure 8. 46,XX DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; CAH: Congenital adrenal hyperplasia; 21OHD: 21α-Hydroxylase deficiency; 11β-OHD: 11β-Hydroxylase deficiency; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; PORD: P450 oxidoreductase deficiency.

*Table 9. Normal Values for AMH (ng/mL) in Fetuses, Newborns and Infants Under 1 Year (122)**
	Female			Male
	n	Median	5-95th percentile	n	Median	5-95th percentile
Fetal blood	25	<0.4	<0.4-0.5	23	44.4	13.4-86.2
Cord blood	53	<0.4	<0.4-1.1	48	30.7	7.4-610.7
1-30 days	70	<0.4	<0.4-1.3	73	57.2	23.8-124.0
31-120 days	31	0.8	<0.4-6.4	39	88.4	46.8-173.0
4-12 months	13	1.0	<0.4-2.7	53	124.7	67.4-197.0

* AMH/MIS enzyme immunoassay kit cat 2368, Immunotech-Beckman Coulter, France

Table 10. LH and FSH Values (means ± SD) From Female and Male Newborns. Modified from (123)
		Female			Male
Age (days)	Hormone	n	Median	Mean±SD	n	Median	Mean±SD
1-5	FSH (IU/L)	31	1.80	2.00 ± 1.37	30	0.85	0.96 ± 0.60
1-5	LH (IU/L)	31	0.20	0.48 ± 0.66	30	0.20	0.39 ± 0.48
6-10	FSH (IU/L)	17	1.40	2.44 ± 2.52	15	1.40	2.91 ± 4.38
6-10	LH (IU/L)	17	0.30	0.45 ± 0.33	15	0.30	0.45 ± 0.33
11-15	FSH (IU/L)	8	8.95	8.16 ± 4.27	17	3.00	3.71 ± 2.69
11-15	LH (IU/L)	8	1.60	1.58 ± 1.28	17	2.90	3.55 ± 2.84
16-20	FSH (IU/L)	6	1.90	1.62 ± 1.05	14	2.15	2.63 ± 1.45
16-20	LH (IU/L)	6	0.35	1.03 ± 1.39	14	3.65	4.13 ± 2.76
21-25	FSH (IU/L)	3	3.90	7.07 ± 5.92	7	2.10	2.50 ± 1.51
21-25	LH (IU/L)	3	0.50	0.46 ± 0.25	7	2.70	2.86 ± 1.51
26-28	FSH (IU/L)	8	6.15	9.74 ± 9.89	8	2.40	2.25 ± 0.81
26-28	LH (IU/L)	8	2.80	2.75 ± 2.39	8	1.40	2.22 ± 2.37

Abbreviations: LH, luteinizing hormone; FSH, follicle-stimulating hormone.

46, XY DSD

A 46, XY karyotype reveals that one is dealing with a genetic male who was under-masculinized during fetal development. A flowchart for differential diagnosis is given in Figure 9.

Laboratory findings of normal or elevated testosterone and DHT with normal AMH (Figure 9) indicate a diagnosis of AIS. If testosterone levels are normal but DHT levels are low, a diagnosis of steroid 5α-reductase deficiency can be made. Low levels of testosterone and DHT, along with marked elevation of some androgen precursors, indicate a deficiency of one of the enzymes required for androgen biosynthesis (Figure 9). For example, if the elevated precursors include androstenedione and 17-hydroxyprogesterone, then the defective enzyme is 17-ketosteroid reductase (HSD17B3). In all cases of testosterone biosynthetic defects, AMH levels are similar to those observed in healthy 46,XY male infants with male-typical external genitalia (Figure 9). With early defects of steroidogenesis that affect both adrenal and gonadal function, it is though important to evaluate adrenal function, as it might be impaired with deficiencies of STAR, CYP11A1, HSD3B2, CYP17A1 and POR (Table 5, Figure 9).

Finally, when all androgens and their precursors are below normal levels, one is dealing with gonadal dysgenesis or 46,XY ovo-testicular DSD. In these cases, AMH values should also be low. In contrast, for babies affected by Leydig cell hypoplasia, androgens and their precursors are low, while AMH values should be in the normal male range.

Figure 9. 46,XY DSD decision tree diagram.
Abbreviations: USG: Ultrasound; 17OHP: 17OH-Progesterone; DSD: Differences in sex development; AMH: Anti-Müllerian hormone; T: Testosterone; N: Normal; PMDS: Persistent Müllerian duct syndrome; AIS: Androgen insensitivity syndrome; SRD5A2: 5α-reductase A2; HCG: Human chorionic gonadotropin; DHT: Dihydrotestosterone; 3β-HSD2: 3β-Hydroxysteroid dehydrogenase type 2; CYP17A1: 17α-Hydroxylase/17-20 lyase; POR: P450 oxidoreductase; StAR: Steroidogenic acute regulatory protein; CYP11A1; Cholesterol side-chain cleavage enzyme; 17β-HSD3: 17β-Hydroxysteroid dehydrogenase type 3; AKR1C2/4: Aldo-keto reductase family 1 member C2/4; CYB5: Cytochrome b5.

Testing Early Postnatal Gonadal Function and Mini-Puberty

In cases of ambiguous genitalia where a diagnosis may not be reached through first line investigations, and where a gonadal malfunction is suspected, functional testing and/or re-analysis of gonadal function at the timepoint of mini-puberty may be helpful, especially also with respect to having information when making considerations towards sex of registration (e.g., in utero effect of sex hormones on the brain) and prediction of later pubertal development and fertility potential.

Mini-puberty is the time window at day 30-100 after birth, when the hypothalamic-pituitary-gonadal (HPG) axis is re-activated for a short time frame in infancy, before going to inactivity until puberty. Little is known about the purpose of mini-puberty, but as the HPG axis is active and stimulates the gonads, it is a time window of opportunity to assess gonadal function without the necessity to stimulate with human chorionic gonadotrophin (hCG).

As a principle, in mini-puberty, the typical ovaries of a 46,XX newborn show little functional activity, while the typical testes of a 46,XY newborn are quite (re)active in testosterone (T), DHT, AMH and inhibin B production. While androgens are produced in the Leydig cells of the testes, AMH and inhibin B originate from the Sertoli cells. Table 11 gives an overview of the hormonal values observed during normal mini-puberty in healthy 46,XX and 46,XY infants (124).

Table 11. Evaluation of Reproductive Hormones During Mini-Puberty in Infants. Normative Values of Different Serum Hormones in 46,XX Female and 46,XY Male Infants. Modified from (124)
		Females			Males
	Post-natal time (months)	n	GMean	2.5-97.5th percentile	n	GMean	2.5-97.5th percentile
LH (IU/L)	2-3.5	432	<LOD	<LOD-0.98	581	1.71	0.62-4.08
LH (IU/L)	3.5-5	110	<LOD	<LOD-1.25	166	1.40	0.54-3.32
FSH (IU/L)	2-3.5	435	3.98	1.23-17.4	578	1.19	0.41- 3.02
FSH (IU/L)	3.5-5	111	3.93	1.30-17.7	165	1.11	0.42-2.68
Testosterone (RIA,nmol/L)	2-3.5	74	<LOD	<LOD-0.40	592	3.04	0.69-7.60
Testosterone (RIA,nmol/L)	3.5-5	14	<LOD	<LOD-NA	168	1.97	<LOD-7.01
Testosterone (LC-MS/MS, nmol/L)	2-3.5	165	<LOD	<LOD-0.21	251	4.75	1.35- 11.3
Testosterone (LC-MS/MS, nmol/L)	3.5-5	125	<LOD	<LOD-0.17	175	2.25	0.32- 9.65
Estradiol (pmol/L)	2-3.5	455	29	<LOD-79	571	<LOD	<LOD-47
Estradiol (pmol/L)	3.5-5	113	31	<LOD-98	160	<LOD	<LOD-49
SHBG (nmol/L)	2-3.5	427	133	67-264	579	135	66-268
SHBG (nmol/L)	3.5-5	110	141	72-282	162	143	71-262
Inhibin B (pg/mL)	2-3.5	423	62	<LOD-184	571	379	229-631
Inhibin B (pg/mL)	3.5-5	106	67	<LOD-174	158	379	222-662
AMH (pmol/L)	2-3.5	339	11	<LOD-49	48	1013	425- 1810
AMH (pmol/L)	3.5-5	67	15	2-46	12	1183	797-NA

Abbreviations: LOD, Limit of detection; GMean, geometric mean; LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone; RIA, radioimmunoassay; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NA, not available.

HCG Testing of Gonadal Function in a DSD Newborn

The human chorionic gonadotrophin (hCG) test can assess whether in a newborn with ambiguous genitalia, functioning Leydig cells are present. The test may therefore be useful for diagnosing testosterone biosynthesis defects such as HSD17B3 or SRD5A2 deficiency (Table 12). Several protocols for the hCG test exist, but all baseline serum samples are taken for T, DHT and precursors. Then, one to three intramuscular injections of high dose hCG (500-1500 IU) are given at 24 hours interval, and finally repeat stimulated samples are taken for androgen measurements after 72 or 24 hours, respectively, after the last injection. The following Table 12 shows how results of the test may be interpreted in various DSD conditions that may lead to ambiguous genitalia (125, 126).

Table 12. Interpretation of hCG Stimulation Test Results (125, 126)
Indication	Consideration	Abnormal result
Testosterone biosynthesis defects	T before and after hCG administration	T increase < 100-150 ng/dL (3.5-5 nmol/L)
5α-Reductase deficiency	T/DHT ratio after hCG administration	≥ 8.5 for minipuberty, ≥ 10 for prepuberty, ≥ 17 for puberty, > 20 in genetically proven cases using LC-MS/MS
17β-HSD3 deficiency	T/A4 ratio after hCG administration	< 0.8
AIS	T, SHBG before and after hCG administration	T increases but SHBG does not change significantly

Abbreviations: T, testosterone; DHT, dihydrotestosterone; HCG, human chorionic gonadotropin; A4, androstenedione; SHBG, sex-hormone binding globulin; AMH, anti-Müllerian hormone; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 17β-HSD3 deficiency, 17β-hydroxysteroid dehydrogenase type 3 deficiency; AIS, androgen insensitivity syndrome

RISK OF MALIGNANCY OF THE DSD GONAD

Individuals with certain disorders of sex development (DSD), especially those involving Y chromosome material, have an increased risk of developing gonadal tumors. These tumors are mainly gonadal germ cell tumors (GGCTs), which occur in about 15 % of all DSD cases, with the risk varying depending on the age and specific underlying diagnosis. Gonadal stromal tumors and smooth muscle leiomyomas/hamartomas are less common and are found in only about 0.9% of DSD cases (127). Risk factors for development of gonadal malignancy in DSD are shown in Figure 10. Several factors contribute to the increased risk of gonadal malignancy in individuals with DSD, including genetic factors, the presence of Y chromosome material, and the type of DSD. Therefore, ongoing monitoring and appropriate management are essential to minimize this risk. While gonadectomy (surgical removal of the gonads) is not recommended for every case of 46,XY DSD, it's crucial to consider the risk of malignant tumors in the decision-making process (1, 69). The consensus statement on the management of DSD provides guidelines and recommendations regarding the timing of gonadectomy for specific patients. These recommendations aim to balance the potential benefits of gonadectomy with the associated risks and individual patient needs.

In individuals with DSDs, germ cells typically undergo a process of apoptosis (programmed cell death) as the body grows and develops. This process eliminates germ cells that are not needed for reproduction. However, in some cases, certain germ cells may not undergo the normal maturation process and remain in an immature state. These immature germ cells can continue to express embryonic markers such as octamer-binding transcription factor 3/4 (OCT3/4) encoded by POU5F1; stem cell factor (SCF), also known as c-KIT ligand (encoded by KITLG); and placental alkaline phosphatase (PLAP). These cells are susceptible to developing into cancerous lesions, starting as pre-invasive lesions (gonado-blastoma) or as precursors to invasive GGCT (dysgerminoma) (Figure 10).

DSD patients who have Y chromosome material in their gonadal karyotype are at risk of GGCTs regardless of the degree of testicular differentiation. Y chromosome material associated with gonadal tumors is GBY locus which is around the centromere of the Y chromosome and includes the gene for ¨testis-specific protein on the Y chromosome¨ (TSPY, located in Yp11.2). Other potentially related genes are SRY and DYZ3.

The risk of developing gonadal tumors in gonadal dysgenesis may also vary according to the underlying molecular etiology. Patients with genetic defects that lead to early blockage of gonadal development such as mutation or deletions of SRY or WT1 have a higher risk. The risk of developing gonadal tumors ranges from 40% to 60% in gonadal dysgenesis due to WT1 gene mutations (Frasier syndrome and Denys-Drash syndrome). Early bilateral gonadectomy is therefore recommended. Similarly, early bilateral gonadectomy is recommended in patients with complete or partial gonadal dysgenesis due to SRY gene mutations due to the 20% to 52.5% risk of gonadal tumor development. Gonadal dysgenesis due to MAP3K1 gene mutations is another indication for early gonadectomy (127). Gonadal tumor risk is also present, though somewhat lower in gonadal dysgenesis due to 45,X0/46,XY mosaicism. The exact time of malignancy development and the prevalence of tumors in other molecular etiologies require further research for a comprehensive understanding.

Histologically the risk of gonadal tumor development increases with gonadal immaturity. Fully developed gonads such as the testes, ovary or ovotestis have lower risk of malignancy than streak or dysgenetic testes (Figure 10). In ovo-testicular DSD, the risk of germ cell tumors is low (3%) (1). Newborns with PAIS are at high risk (50%) for developing gonadal tumors, and bilateral orchiopexy or gonadectomy is recommended at the time of diagnosis, if the testes are located in the abdomen (1, 128).

The risk of developing germ cell malignancy in newborns with CYP11A1, HSD3B2 or CYP17A1 deficiency is unknown. Newborns with 17-ketosteroid reductase deficiency have a fairly low risk for malignancy (1). Tumor risk in newborns with Leydig cell hypoplasia is unclear, and thus the recommendation for gonadectomy is not well established (1, 25, 129, 130).

Overall, it remains challenging to predict the personal risk of gonadal neoplasia (mostly GGCT) for a DSD individual, because it depends on many other factors besides genetics. Stimulating gonadotropins and gonadal hormones during mini-puberty and after puberty onset are also regarded as risk factors increasing gonadal tumor risk in patients with DSD. Similarly, gonads located intra-abdominal carry a higher tumor risk. The management of gonadal tumor risk in individuals with DSD is a complex issue that requires careful consideration and a personalized approach, taking into account the specific DSD subtype, genetic factors, and patient preferences. Regular monitoring and discussions with a healthcare team experienced in DSD management are essential to make informed decisions about gonadectomy. If gonads are located in the scrotum, then periodic examination of the testes may be advised as an alternative to gonadectomy, while an intra-abdominal gonad at risk may only be monitored by imaging methods such as ultrasound or MRI.

Figure 10. Risk of malignancy development in the DSD gonad.
Abbreviations: (OCT3/4); Octamer-binding transcription factor 3/4, TSPY; testis-specific protein on the Y chromosome, c-KITL; c-KIT ligand also called stem cell factor (SCF).

SEX REGISTRATION OF A NEWBORN

Assigning or registering the sex of a newborn with atypical external genitalia is a complex and important decision that requires careful consideration, consultation, and a patient-centered approach. The parents and DSD network team should not rush but take the time needed to come to a consensus in the best interest of the child in its environment. Not knowing whether the newborn is a girl or boy causes mostly a lot of distress and one is tempted to call it an emergency, but if there are no medical reasons to make it an emergency (like e.g., suspected adrenal insufficiency or additional organ anomalies), it is important to declare it accordingly and inform that the newborn is not at risk and actually does not care whether it is a male or female or intersex person at this moment. Thus, the stress of ambiguity of the newborn is actually at this moment “only” an issue of the parents and the care team who mostly wish to know ASAP whether the child can be assigned/registered a “correct” sex that might also fit later in life.

To consider sex registration, not only is it important to take into account the underlying cause and knowledge on the possible outcome of the specific, often unique condition, but also the family, cultural, and societal preconditions and aspects. Most societies have a binary sex registration obligation by legislation, but some societies have more recently introduced a third sex category besides male and female. Although this seems to make things easier, it has been discussed that such categorization in a third or other or unclassified group might also stigmatize a person in a society that is mostly binary. Thus, in real life practice, there is rumor that a third sex category is rarely chosen for a newborn with ambiguous genitalia by the parents and the DSD team. Overall, a personalized approach for every newborn with ambiguous genitalia involving the parents and the DSD network team is recommended following the shared-decision-making process (12, 13).

Most important is that the team around the newborn with ambiguous genitalia works together in the best interest of the child with the latest knowledge of the literature and expertise when taking the decision of sex registration, and still remains humble and open minded for the child’s future that may follow a different path leading to sex reassignment later in life. Therefore, continuous support of the family and the child by a DSD psychologist is extremely helpful to recognize early signs of gender dysphoria, inform the child about the condition in an age-appropriate manner and follow its will, if case sex reassignment is desired.

Generally, people believe that the sex chromosomes indicate a person’s “true sex” and laws exist supporting this idea, to the detriment of some individuals affected by DSD (131). Scientifically speaking, it is clear that the majority of genes on the X chromosome do not influence sex development and differentiation, although the AR gene is necessary for phenotypic masculinization. Concerning the Y chromosome, only the SRY gene contributes to testicular formation. In fact, most of the genes required for sex development and differentiation are found on the autosomes (1). Therefore, chromosomes do not dictate the sex of rearing in newborns affected by a DSD and are only one piece of a big puzzle (Figure 3).

Although there is no overall recommendation for sex registration at birth for a child with ambiguous genitalia, some helpful information is available from larger patients’ cohorts with a specific DSD diagnosis in the literature.

Newborns presenting with 46,XY chromosomes and female external genitalia at birth (thus actually not with ambiguous genitalia) due to complete androgen insensitivity syndromes (CAIS), complete gonadal dysgenesis (Swyer syndrome), or other complete loss of testosterone biosynthesis live successfully when assigned a female sex of rearing. Female assignment in such cases is widely accepted by patients throughout their lives despite challenges that they may experience regarding sexual dysfunction and infertility (1, 132-134).

For newborns with ambiguous genitalia affected by partial gonadal dysgenesis, no general recommendation can be made. Fewer procedures were historically required for surgical feminization compared to surgical masculinization of the genitalia. However, the functional outcome in individuals who received feminizing or masculinizing procedures is less than optimal (135, 136). Additionally, rates of satisfaction with sex of rearing are similar for affected individuals whether raised male or female (1, 20, 137). Thus, the decision should not be made on the basis of “surgical possibilities”, and overall, such surgical procedures should be executed with reservation. Clearly, further studies are needed to elucidate why some individuals with partial gonadal dysgenesis experience gender dysphoria, while others do not.

Likewise, no general recommendation can be made for patients with ambiguous genitalia due to a partial androgen biosynthesis defect, (e.g., partial 3βHSD2, CYP17A1, POR, StAR, CYP11A1 deficiencies). But some studies suggest that the degree of masculinization of the external genitalia may reflect the degree of virilization of the brain and thus the gender preference of this person (138). This might be true for 46,XY newborns with ambiguous genitalia that are manifesting with severe hypospadias, but not without exceptions. For patients with variants in the SRD5A2 or HSD17B3genes recognized with ambiguous genitalia at birth (or even missed because manifesting with a female-typical external genitalia), there is quite a strong body of data recommending male sex registration at birth as this is the preferred sex of these persons when asked in later life (139, 140). Normal virilization at puberty, coupled with intact fertility potential, are strong factors for this recommendation at birth. If registered as female at birth and diagnosed later, gender dysphoria and desire of sex reassignment later in life is very likely.

Similar to partial gonadal dysgenesis, consensus regarding an optimal sex of registration for newborns affected by PAIS does not exist. Although earlier data showed that fewer procedures were usually required for surgical feminization, compared to surgical masculinization, in a person with ambiguous external genitalia in the past, when larger series of genital surgery were analyzed, it is unclear if the functional outcome was better among newborns who received feminizing procedures compared to those who received masculinizing procedures. Rates of satisfaction with sex of rearing are similar for individuals with PAIS raised female or male (132, 141). While reports exist of impaired sexual function in people with PAIS raised male, it is highly suspected that sexual functional outcomes in affected people raised female are similarly poor (137). Reports of sexual satisfaction for people with PAIS who have not received genitoplasty in infancy, or at a later age, are limited. However, more information on outcome of newborns with ambiguous genitalia who had no genital surgery, at least not in infancy and childhood, will hopefully soon be added to the medical literature as current developments recommend abstaining from genital surgery.

By contrast, in 46,XX newborns with ambiguous genitalia due to CAH, it is generally recommended to register them a priori in the female sex, even if they are severely virilized (142-144). Masculinization due to 21-hydroxylase deficiency does not impair the development of the ovaries or the Müllerian ducts and thus fertility potential. Limited information is available about the medical and psychosexual outcome of 46,XX newborns affected by 21-hydroxylase deficiency and raised male (145, 146), although these patients do tend to have an increased risk of gender dysphoria compared to their female counterparts (147). Thus, a female sex of rearing is typically the decision for 46,XX newborns affected by 21-hydroxylase deficiency (145). Female sex of rearing in 46,XX newborns with ambiguous genitalia is also recommended for conditions resulting from maternal overexposure to androgens and placental aromatase deficiency.

FINAL REMARKS TO OVERALL MANAGEMENT

Only recently has the understanding of, and reaction to, having a child with ambiguous genitalia received systematic study. For some parents and caregivers, feelings of isolation and concern over what the future may hold for their affected child in terms of stigmatization and sexual dysfunction are paramount (148). One potential approach to ameliorate this issue is to provide information and management options to the family early in pregnancy or as soon as the child is born. Guidance by a DSD network team is therefore extremely important.

Some research has been done to examine the effects of prenatal diagnosis and treatment. In females with 21-hydroxylase deficiency CAH, one of the most common causes of ambiguous genitalia, dexamethasone treatment of the pregnant mother has been reported to reduce virilization of an affected female fetus by 80-85% (149), but this approach is no longer offered or recommended in guidelines because of potential adverse effects of dexamethasone on the developing fetus. Before the discovery of methods to measure fetal DNA in maternal blood, which enabled specific diagnosis of sex and whether the fetus inherited and was affected by the mutation, controversies existed because 7 unaffected fetuses would be treated unnecessarily (1:4 chance of inherited mutation and 1:2 chance of male vs female), So, on the one hand 7 unaffected fetuses and the mother would be exposed to an unnecessary drug, while on the other hand, even in affected fetuses long-term outcome showed adverse effects on neurocognitive parameters (150). Today, noninvasive prenatal diagnosis using massive parallel sequencing of cell-free fetal DNA can be used to determine a specific diagnosis, such as CAH, as early as 6 weeks of gestation (149, 151). Even preimplantation diagnostics is available (152). These techniques could be used to look at other autosomal dominant or X-linked conditions that cause ambiguous genitalia, especially when the fetus is known to be at risk (151). Further studies need to be done on this subject, but for families who already have an affected child or know that they are carriers, this may be an option that needs to be discussed with a DSD geneticist.

Patients for whom prenatal diagnosis and/or treatment is not an option, prognosis and management has improved significantly over time, but many open questions and controversies still exist. Our studies of parents of children with DSD reveal that the appearance of atypical genitalia can result in significant stress and maladaptive parenting strategies. For example, mothers of children with ambiguous genitalia experience greater stress if their child has not received “corrective” genital surgery (153) and some parents believe such surgeries eliminate stigmatization that may arise due to their child’s ambiguous genitalia However, as their children mature past infancy, some parents realize that their child’s DSD has not been ameliorated by genitoplasty, and concerns for their son or daughter resurface (148). To illustrate, among parents studied by our group, caregivers of adolescents with DSD experienced increased stress as their child matured despite the fact that many had received earlier surgery (154). Additionally, parents of children reared male reported the most perceived child vulnerability for their sons (155),and their reported levels of depression were directly associated with more atypical genital appearance for their sons (156). Overall, parents report that while early genitoplasty seems to “fix” some of their concerns for their child with DSD, this “fix” is not long-lasting. Instead, what needs to be emphasized to families is how to support their child who is different, but not damaged. Family centered, interdisciplinary care, with open and clear communication between patients, parents, and caregivers has been shown to be essential for optimal quality of life (157). In addition, networks of families who have the personal experience of parenting children with ambiguous genitalia can be an invaluable resource (158). Reservation towards early interventions needs to be considered,keeping in mind to preserve the personal right of the child to take his/her own decision towards body integrity. Any intervention that can be delayed without harming the newborn with ambiguous genitalia should be postponed until personal consent can be given. Finally, referring parents to support groups and introducing them to other caregivers of children born with ambiguous genitalia is extremely important to optimize parents’ understanding and acceptance of their child’s condition. With increased understanding and acceptance, optimal growth and development for children born with ambiguous genitalia may be obtained.

In conclusion, this Chapter give some insight into the current diagnostic evaluation and care of the newborn with ambiguous genitalia. We underscore the importance of a multidisciplinary and compassionate approach to the care of newborns with ambiguous genitalia, acknowledging the complex medical, psychological, and emotional aspects involved in such cases. We highlight the critical role of specialized teams and support in ensuring the well-being of both the child and his/her family.

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Fibromyalgia

ABSTRACT

Fibromyalgia is a clinical entity characterized by the combination of chronic widespread pain and other non-pain symptoms, including fatigue, poor sleep, and cognitive disturbances, which can exhibit symptom variation not only between different patients, but also in the same patient during the course of the disease. These symptoms are relatively common and non-specific. They can be encountered in other disorders that may overlap with fibromyalgia, often without having clear boundaries, while their nature makes them difficult to be objectively defined and quantified. These issues have led to significant controversy over the definition and the diagnostic criteria of fibromyalgia. It has been suggested that the markers of physical and psychological distress have a continuous distribution in the general population with fibromyalgia patients being at the extreme end of this continuum. Genetic predisposition in combination with environmental factors, are responsible for each individual’s position in this this distribution. In recent years more knowledge has been obtained to better understand the environmental factors that seem to be important in triggering fibromyalgia. Most of them act as stressors superimposed onto a deranged stress-response system leading to dys-regulation of the nociceptive system and the appearance of clinical symptoms. The aim of the therapy is to relieve pain and motivate the patients to become more physically active using a multimodal individualized therapeutic strategy that includes education, exercise, cognitive-behavioral approaches and medications. The response to current therapeutic modalities varies significantly, with some patients responding adequately, while others do not seem to experience any long-term benefit.

INTRODUCTION

Fibromyalgia is a clinical entity characterized by the combination of ill-defined symptoms including chronic widespread pain, with concomitant fatigue, sleeping disorders, and cognitive disturbances (1). The severity of these symptoms can vary significantly during the course of the disease. Fibromyalgia has been described as an arbitrarily created syndrome that lies at the extreme end of the spectrum of poly-symptomatic distress (2). The term poly-symptomatic was used to emphasize the variety of multiple different symptoms that can be found in fibromyalgia patients, while the distress can have a physical and/or a psychological component. This exact nature of fibromyalgia makes it difficult to be clearly defined, often overlapping with disorders that are characterized by similar symptoms. It is important to note that fibromyalgia is not an exclusion diagnosis as it can co-exist with other clinical conditions (3).

CLINICAL FEATURES

The main presenting complains of patients with fibromyalgia include chronic widespread pain (also called multisite pain), fatigue, and poor sleep. Usually the pain is initially localized, but eventually it involves many muscle groups. It is characterized as persistent with varying intensity, while it can often be described as a sensation of burning, gnawing soreness, stiffness, or aching. Excessive sensitivity to normally painful stimuli, such as pressure or heat (hyperalgesia) and painful sensation to normally non-painful stimuli, such as touch (allodynia) are significant features of fibromyalgia. Often patients complain of swollen joints and paresthesias without though the presence of any objective clinical findings during physical examination. Pain is often aggravated by cold and humid weather, poor sleep, physical and mental stress. Additionally the patients may have a variety of less well understood pain symptoms, including abdominal pain, chest wall pain, symptoms suggestive of irritable bowel syndrome, pelvic pain, and bladder symptoms of frequency and urgency suggestive of interstitial cystitis (4–9).

Fatigue is present in almost all patients with fibromyalgia, while many complain of non-refreshing sleep, frequent awakening during the night, and difficulty falling back to sleep. Sleep apnea and nocturnal myoclonus can also be present along with a sensation of light-headedness, dizziness, and faintness. In addition, cognitive difficulties such as short-term memory loss, groping for words and poor vocabulary, are common among patients with fibromyalgia. Mood disturbances, including depression, anxiety and heightened somatic concern, may often also occur. Headaches, either muscular or migraine type, are also commonly present (6,7). Other often co-existing conditions include multiple chemical sensitivity, “allergic” symptoms, ocular dryness, palpitations, dyspnea, vulvodynia, dysmenorrhea, premenstrual syndrome, sexual dysfunction, weight fluctuations, night sweats, dysphagia, restless leg syndrome, temporomandibular joint pain, chronic fatigue syndrome (systemic exertion intolerance disease), Raynaud`s phenomenon, autonomic dysfunction, and dysgeusia (6,8,9). These conditions cannot be used to support the diagnosis of fibromyalgia.

Approximately 40% of fibromyalgia patients have accompanying depression at the time of diagnosis, while 60% of patients have a lifetime history of depression. In addition, an anxiety disorder is present in 30% of the cases at the time of diagnosis while the lifetime prevalence of an anxiety disorder in fibromyalgia patients is approximately 60% (10–14). The levels of depression and anxiety in patients with fibromyalgia seem to be associated with the degree of cognitive impairment, as shown in a meta-analysis of 23 case-control studies (15). Based on the coexistence of depression and anxiety, fibromyalgia patients can be divided into 2 major groups. The first group comprises of patients without coexisting mood disorders, while the second of patients with concomitant depressive mood, often in combination with anxiety. According to the results of a study that intended to subgroup fibromyalgia patients based on: 1) mood status (evaluated by the Center for Epidemiologic Studies Depression Scale for depression and the State-Trait Personality Inventory for symptoms of trait-related anxiety), 2) cognition (by the catastrophizing and control of pain subscales of the Coping Strategies Questionnaire), and 3) hyperalgesia/tenderness (by dolorimetry and random pressure-pain applied at suprathreshold values), it was noted that fibromyalgia patients with depressive mood and anxiety are also ‘catastrophizing’. This term is used to indicate that such patients have a very negative, pessimistic view of what their pain is and what is causing, while they have no sense that they can control their pain. On the contrary fibromyalgia patients who are neither depressed nor anxious and therefore do not catastrophize, have a moderate sense that they can control their pain. These patients can be further divided into 2 subgroups based on the degree of hyperalgesia/tenderness, the first subgroup comprises of patients with high hyperalgesia/tenderness, while the second one of patients with moderate hyperalgesia/tenderness (11). In addition, it has been proposed that depressed fibromyalgia patients can also be divided into 2 subgroups, in the first one depression is a co-morbid condition, while in the second depression is the cause of fibromyalgia (16). All these fibromyalgia subgroups are illustrated in Figure 1.

Figure 1. Subgroups of fibromyalgia patients.

In another study fibromyalgia patients were classified as dysfunctional, inter-personally distressed, or adaptive copers, based on their responses to the Multidimensional Pain Inventory. The dysfunctional patients experienced more pain behaviors and overt expressions of pain, distress, and suffering, such as slowed movement, bracing, limping, and grimacing compared to the inter-personally distressed or the adaptive copers (17).

It is of interest that up to 25% of patients correctly diagnosed with a systemic rheumatic disease (e.g. rheumatoid arthritis, systemic lupus erythematosus) will also fulfill the classification criteria for fibromyalgia (18). This is also the case for many patients who experience persistent various forms of pain (including widespread myalgias, arthralgias, and headache), fatigue, neurocognitive dysfunction, and sleep disturbances after an infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that caused a mild to moderate coronavirus disease 2019 (long COVID) (19,20). The most commonly encountered comorbid conditions in fibromyalgia patients are shown in Table 1.

- Table 1. The Most Commonly Encountered Co-Morbid Conditions in Fibromyalgia
Sleep disorders	- Non restorative sleep (alpha-delta sleep anomaly) - Sleep apnea - Restless leg syndrome - Nocturnal myoclonus
Chronic fatigue syndrome (systemic exertion intolerance disease)
Psychiatric disorders	- Anxiety disorders - Depression - Obsessive compulsive disorder
Headache	- Tension type headache - Migraine
Irritable bowel syndrome
Musculofascial pain syndrome	- Temporomandibular disorders - Interstitial cystitis
Dysmenorrhea
Premenstrual syndrome
Non-cardiac chest pain
Raynaud’s phenomenon
Systemic autoimmune diseases	- Rheumatoid arthritis - Systematic lupus erythematosus - Sjögren’s syndrome - Ankylosing spondylitis and other seronegative spondylarthritis - Polymyalgia rheumatica
Long COVID

ASSESSMENT

Fibromyalgia is a chronic illness, with a variety of symptoms that can change during the course of the disease and after treatment. Therefore, its core symptoms should be evaluated both in clinical practice and in treatment trials. A working group within OMERACT (Outcome Measures in Rheumatology) reached a consensus regarding the domains that need to be assessed in clinical trials for fibromyalgia, using Delphi exercises within patients and expert clinicians (21). The fibromyalgia core symptom domains include pain intensity, tenderness, fatigue, sleep disturbance, multidimensional function (including health related quality of life and physical function), patient global impression of change, cognitive dysfunction, and depression. However at the present time, there is no consensus on how to evaluate these domains, in order to quantify fibromyalgia disease activity state and/or response (22).

Pain intensity can be assessed using visual analog scales. It has been proposed to use the wording “please rate your pain by circling on the number that best describes your pain on average” and has anchors that vary from “no pain” to “pain as bad as you can imagine” (23).

Tenderness can be measured by evaluating the alteration of the severity of pain at the tender points, using visual or analog scales, but not by the change in their number. The change in the number of tender points is poorly correlated with improvement in fibromyalgia treatment trials. It has been noted that the tender points measure the combination of tenderness and distress an individual has, rendering them inadequate for the evaluation of tenderness per se (24).

Fatigue can be evaluated by the Fatigue Severity Scale (FSS), which measures the functional outcomes related to fatigue (25). It has been shown that FSS has the most robust psychometric properties of 19 reviewed fatigue measures, while it had the best ability to act as an outcome measure sensitive to change with treatment, in chronically ill patients (26). Other instruments that have been used to assess fatigue in fibromyalgia patients include the Multidimensional Fatigue Index and the Multidimensional Assessment of Fatigue.

Sleep disturbance in fibromyalgia patients has been evaluated by the Medical Outcome Studies (MOS) sleep scale, the Functional Outcomes of Sleep Questionnaire (FOSQ), and the Jenkins Sleep Scale (JSS). Of these instruments MOS sleep scale lacks validity to assess changes in sleep symptoms in fibromyalgia treatment trials, FOSQ has not been adequately validated in fibromyalgia patients, while JSS has been criticized for possible high-recall bias, since it requires the patients to rate the frequency of their symptoms over a period of a month (23).

Multidimensional function, including health related quality of life and physical function, can be evaluated using the fibromyalgia impact questionnaire (FIQ). This represents a useful tool in assessing functional abilities in daily life and measures patient status, progress and outcomes. FIQ is self-administered and is highly sensitive to changes during the course of the disease. However, its functional items are orientated toward high levels of disability, resulting in a possible floor effect, while its physical function items are addressed to women living in affluent countries, generating gender and ethnic bias. To address these issues the Revised Fibromyalgia Impact Questionnaire (FIQR) was developed, having modified physical function questions and including questions on memory, tenderness, balance and environmental sensitivity, while keeping questions that evaluate overall impact and symptoms (27). Other tools for assessing overall function and quality of life include the Health Assessment Questionnaire, the Symptom Interpretation Questionnaire, the Western Ontario and McMaster Universities Osteoarthritis Index, the Patient Global Impression of Change scale (PGIC), and psychometric scales (28).

Patient global impression of change can be evaluated by the Patient Global Impression of Change scale (PGIC), as recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) (29).

Cognitive dysfunction can be evaluated by the Multiple Ability Self-report Questionnaire (MASQ), which is a self-report questionnaire measuring language, visuoperception, verbal memory and attention (30). However self-assessment is poorly correlated with objective measures of cognitive function and has poor discriminating ability for patients with mild cognitive impairment (31).

Depression can be evaluated using the Hospital Anxiety and Depression Scale (HADS) depression subscale (HADS-D). The use of the original Beck Depression Inventory (BDI) as well as the current BDI-II, should be avoided, since they tend to overestimate the presence of major depressive disorder in fibromyalgia patients by evaluating a number of non-depressive symptoms, which are often encountered in fibromyalgia (23).

DIAGNOSIS

As already mentioned, fibromyalgia is characterized by symptoms that can vary in number and intensity not only between different patients, but also in the same patient during the course of the disease. These symptoms are also common in other disorders that can overlap with fibromyalgia, often without having clear boundaries. Additionally, the nature of the symptoms of fibromyalgia makes them difficult to be objectively defined and measured. All these issues have led to significant controversy over the definition and diagnosis of fibromyalgia. The clinical entity of fibromyalgia was first described in 1904, under the term “fibrositis”, after focusing on clinical evidence of muscle sensitivity (32). It was not until 1977 that specific criteria for the diagnosis of fibromyalgia were introduced (33). Since then a number of different criteria have been proposed, based on a combination of tender point examination and the presence of symptoms. In 1986 a committee of the American College of Rheumatology (ACR) started a multicenter study, trying to provide a definition of fibromyalgia and establish classification criteria (34). In 2013, the Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks (ACTTION) public-private partnership with the US Food and Drug Administration (FDA) and American Pain Society (APS) initiated the ACTTION-APS Pain Taxonomy (AAPT) in an attempt to develop a diagnostic system that would be clinically useful and consistent across chronic pain disorders, including fibromyalgia (35).

In clinical practice either the 2016 revision of the 2010/2011 fibromyalgia diagnostic criteria or the AAPT criteria can be used to help physicians to diagnose fibromyalgia (36). However, these criteria should be viewed as an aid and not as a gold standard for diagnosing fibromyalgia in clinical practice. Good clinical judgment is necessary to interpret the findings of physical examination and to assess psychological factors and associated comorbidities so as to correctly identify the patients with fibromyalgia (37).

Relevant social, personal and family history can be helpful in establishing the diagnosis of fibromyalgia, since there is evidence that the symptoms of fibromyalgia can appear after a physical or emotional trauma, a medical illness, or asurgical operation, while a family history of fibromyalgia, makes the diagnosis of fibromyalgia more likely (38).

1990 ACR Classification Criteria

In 1990 the American College of Rheumatology (ACR) committee established criteria for the classification of fibromyalgia. According to these criteria fibromyalgia is defined as chronic widespread pain involving both sides of the body, above and below the waist, as well as the whole length of the spine, and excessive tenderness in the pressure of 11 of 18 specific muscle-tendon sites (9 pairs of tender points). The locations of the tender points are described in Figure2 and Table 2. Pressure equivalent of 4 kg/cm is applied to these points using the pulp of the thumb or the first two or three fingers. This can be accurately measured with a dolorimeter or it can be estimated, since 4 kg/cm is the pressure needed to be applied so as to whiten the examiner’s fingernail bed. These criteria specifically state that fibromyalgia is not an exclusionary diagnosis (34).

Figure 2. Fibromyalgia tender points.

Table 2. Description of the Location of Fibromyalgia Tender Points
Occiput:	at the insertions of one or more of the following muscles: trapezius, sternocleidomastoid, splenius capitus, semispinalis capitus
Trapezius:	at the midpoint of the upper border
Supraspinatus:	above the scapular spine near the medial border
Gluteal:	at the upper outer quadrant of the buttocks at the anterior edge of the gluteus maximus
Low cervical:	at the anterior aspect of the interspaces between the transverse processes of C5–C7
Second rib:	just lateral to the second costochondral junctions
Lateral epicondyle:	2 cm distal to the lateral epicondyle
Greater trochanter:	posterior to the greater trochanteric prominence
Knee:	at the medial fat pad proximal to the joint line

The tender points, which are examined in fibromyalgia, are not just areas that the patient feels pain. They are points that fibromyalgia patients are relatively more tender, compared to normal individuals, when pressure is applied on them. But fibromyalgia patients are more tender wherever you apply pressure, not only to some of these 18 specific tender points, including areas previously considered to be “control points” (39). There is evidence that these tender points are areas that everyone is generally more tender. In contrast, fibromyalgia patients are more tender not only to pressure but to other stimuli such as heat, cold and other sensory stimuli, most probably due to decreased pain threshold. The number of tender points an individual has is highly correlated with distress, as defined by the presence of anxiety, depression, sleep disturbance, fatigue and global severity. Tender points have been described as “a sedimentation rate of distress”. Consequently tender points measure the combination of tenderness and distress an individual has (24).

The use of the 1990 ACR classification criteria in clinical practice is surrounded by substantial controversy. Tender points were introduced as an objective physical finding. However, if the physician who performs the physical examination is not experienced enough, tender point counting is impossible to be performed accurately. Most of the physicians examining fibromyalgia patients did not know how to carry out the tender point examination. Consequently, tender point count was not routinely performed, and when performed it was performed inaccurately (40). Another shortcoming of the tender point counting is that it is not as objective as it was initially considered, since the physician can be biased by the patient’s interview that precedes the physical examination. These issues lead to a low inter-examiner reliability of the tender point count (41).

Other more sophisticated measures of assessing tenderness, such as applying stimuli randomly, when the individual cannot anticipate what the next stimulus is going to be, are equally abnormal in fibromyalgia patients, but do not correlate with distress (39). These methods require special training and are more time consuming than the trigger point count. Other alternative assessment methods include functional magnetic resonance imaging (fMRI) and nociceptive flexion reflex (NFR) testing, which documents abnormal pain processing in fibromyalgia. Functional MRI demonstrates similar brain activation in regions involved in pain processing in fibromyalgia patients and normal individuals. However fibromyalgia patients have increased pain sensitivity and brain activation during comparable stimulus (42). Nociceptive flexion reflexes are sensory-motor responses elicited by electrical noxious stimuli, which involve activation of spinal and supraspinal neuronal circuits, providing an objective and quantitative assessment of the function of the pain-controlsystem. It has been demonstrated that the NFR threshold in patients with fibromyalgia is significantly decreased compared with that in controls (43). Of these methods fMRI is expensive and complex compared to NFR testing that appears to be more easily accessible and convenient, since standard electromyographic equipment can be used. This test also seems to eliminate subjective bias and dissimulation (44).

The 1990 ACR classification criteria, define fibromyalgia in terms of pain rather than its other features. However, patients with fibromyalgia apart from tenderness and pain, also have a number of other somatic symptoms. Although non-pain symptoms are important, there is no evidence to support the notion that they are more important than hyperalgesia and allodynia, which are key symptoms of fibromyalgia (44). Many clinicians with experience in fibromyalgia did not feel that the 1990 ACR classification criteria were sufficiently reliable for the diagnosis of fibromyalgia in clinical practice and were considering other aspects of the disease in an attempt to reach a more accurate diagnosis (38).

2010 ACR Preliminary Diagnostic Criteria

To address the aforementioned issues the ACR in 2010 proposed the preliminary diagnostic criteria for fibromyalgia (Table 3), that were not meant to replace the 1990 ACR classification criteria, but to represent an alternative simple and easy method of diagnosis in clinical practice (45). These diagnostic criteria do not require a tender point count. Instead, they rely only on symptoms for the diagnosis of fibromyalgia. They introduced the widespread pain index (WPI), which counts the areas that the patient feels pain during one week preceding the examination, and the symptom severity (SS) scale, which describes the severity of fatigue, unrefreshing sleep, cognitive problems, and a number of associated somatic fibromyalgia symptoms. These symptoms need to be assessed and rated by a physician, therefore the 2010 ACR preliminary diagnostic criteria are in-adequate for patient self-diagnosis.

Two more conditions need to be fulfilled so as to diagnose fibromyalgia. The symptoms need to be present at a similar level for at least 3 months while alternate disorders that would otherwise explain the pain need to be excluded (Table 3). The authors of the 2010 ACR preliminary diagnostic criteria have clarified that the latter condition does not mean that fibromyalgia is an exclusion diagnosis according to these criteria. The diagnosis of fibromyalgia should not be made only when there is not another disease that could explain the pain that would otherwise be attributed to fibromyalgia. It should be noted that rheumatic diseases usually do not cause pain that can be confused with fibromyalgia (3).

Table 3. 2010 ACR Preliminary Diagnostic Criteria
Criteria: A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met: 1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or Widespread pain index (WPI) 3-6 and symptom severity (SS) scale score ≥9. 2) Symptoms have been present at a similar level for at least 3 months. 3) The patient does not have a disorder that would otherwise explain the pain. Ascertainment: 1) WPI Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain? (Score will be between 0 and 19)
	-Neck	-Upper arm, left	-Abdomen	-Upper leg, left
	-Jaw, left	-Upper arm, right	-Upper back	-Upper leg, right
	-Jaw, right	-Lower arm, left	-Lower back	-Lower leg, left
	-Shoulder girdle, left	-Lower arm, right	-Hip (buttock, trochanter), left	-Lower leg, right
	-Shoulder girdle, right	-Chest	-Hip (buttock, trochanter), right
2) SS scale score The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the extent (severity) of somatic symptoms in general. (The final score is between 0 and 12) -For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale: 0 = no problem 1 = slight or mild problems, generally mild or intermittent 2 = moderate, considerable problems, often present and/or at a moderate level 3 = severe: pervasive, continuous, life-disturbing problems Fatigue (0-3) Waking unrefreshed (0-3) Cognitive symptoms (0-3) -Considering somatic symptoms in general, indicate whether the patient has: muscle pain, irritable bowel syndrome, fatigue/tiredness, thinking or remembering problem, muscle weakness, headache, pain/cramps in the abdomen, numbness/tingling, dizziness, insomnia, depression, constipation, pain in the upper abdomen, nausea, nervousness, chest pain, blurred vision, fever, diarrhea, dry mouth, itching, wheezing, Raynaud's phenomenon, hives/welts, ringing in ears, vomiting, heartburn, oral ulcers, loss of/change in taste, seizures, dry eyes, shortness of breath, loss of appetite, rash, sun sensitivity, hearing difficulties, easy bruising, hair loss, frequent urination, painful urination, and bladder spasms 0 = no symptoms 1 = few symptoms 2 = a moderate number of symptoms 3 = a great deal of symptoms

Table 3. 2010 ACR Preliminary Diagnostic Criteria

Criteria:

A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met:

1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or

Widespread pain index (WPI) 3-6 and symptom severity (SS) scale score ≥9.

2) Symptoms have been present at a similar level for at least 3 months.

3) The patient does not have a disorder that would otherwise explain the pain.

Ascertainment:

1) WPI

Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain?

(Score will be between 0 and 19)

-Neck

-Upper arm, left

-Abdomen

-Upper leg, left

-Jaw, left

-Upper arm, right

-Upper back

-Upper leg, right

-Jaw, right

-Lower arm, left

-Lower back

-Lower leg, left

-Shoulder girdle, left

-Lower arm, right

-Hip (buttock, trochanter), left

-Lower leg, right

-Shoulder girdle, right

-Chest

-Hip (buttock, trochanter), right

2) SS scale score

The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the extent (severity) of somatic symptoms in general.

(The final score is between 0 and 12)

-For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale:

0 = no problem

1 = slight or mild problems, generally mild or intermittent

2 = moderate, considerable problems, often present and/or at a moderate level

3 = severe: pervasive, continuous, life-disturbing problems

Fatigue (0-3)

Waking unrefreshed (0-3)

Cognitive symptoms (0-3)

-Considering somatic symptoms in general, indicate whether the patient has: muscle pain, irritable bowel syndrome, fatigue/tiredness, thinking or remembering problem, muscle weakness, headache, pain/cramps in the abdomen, numbness/tingling, dizziness, insomnia, depression, constipation, pain in the upper abdomen, nausea, nervousness, chest pain, blurred vision, fever, diarrhea, dry mouth, itching, wheezing, Raynaud's phenomenon, hives/welts, ringing in ears, vomiting, heartburn, oral ulcers, loss of/change in taste, seizures, dry eyes, shortness of breath, loss of appetite, rash, sun sensitivity, hearing difficulties, easy bruising, hair loss, frequent urination, painful urination, and bladder spasms

0 = no symptoms

1 = few symptoms

2 = a moderate number of symptoms

3 = a great deal of symptoms

There is evidence that there is good agreement between the 1990 ACR classification criteria and the 2010 ACR preliminary diagnostic criteria (46–53). However, these criteria are expected not to agree completely, as the former are focused on the presence of tender points while the latter on the presence of symptoms. The 1990 criteria can diagnose fibromyalgia in patients who do not have sufficiently high symptom score according to the 2010 criteria, while the 2010 criteria can diagnose fibromyalgia in patients who do not have sufficient tender points according to the 1990 criteria.

The introduction of the 2010 ACR preliminary diagnostic criteria was surrounded by controversy too. In particular, they have been criticized for being completely symptom focused, ill-defined, and lucking some mechanistic features of fibromyalgia, such as hyperalgesia, central sensitization and dysfunctional pain modulation (54). Additionally, these diagnostic criteria are based on the subjective assessment of the patient’s somatic symptoms by the physician, adding ambiguity and influencing repeatability among different physicians (55). A self-reported version of the 2010 ACR preliminary diagnostic criteria was developed in 2011, so as to be used in survey research, and not in clinical practice (56). These criteria are known as the modified 2010 ACR preliminary diagnostic criteria or the 2011 ACR survey criteria. They introduced the fibromyalgia severity (FS) score (originally called fibromyalgianess scale) which is the sum of the self-reported WPI and SS score. This score can be used as an approximate measure of the severity of fibromyalgia. The FS score has also been called polysymptomatic distress (PSD) scale. It has been proposed that the markers of physical and psychological distress have a continuous distribution in the general population with fibromyalgia patients being at the extreme end of this distribution (57). The PSD scale could be useful to define the position of each individual in this continuum, without having to differentiate between patients with fibromyalgia and those without, as this distinction can sometimes be unclear if not arbitrary (58).

2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria

A limitation of the WPI is the fact that it counts the number of painful areas without considering their distribution in the body. Patients with regional pain disorders can fulfill the 2010 ACR preliminary diagnostic criteria since pain can be located in 3 or more areas in the same region (59). To overcome this issue the 2016 revision of the diagnostic criteria require the pain to be generalized (multisite pain). The areas WPI assesses are divided in 5 regions (Table 4) and the diagnosis of fibromyalgia requires the distribution of pain in 4 out of 5 regions (60). The jaw, the chest and the abdomen area are problematic when they are used to define a region. In this way they are excluded from the definition of generalized pain (61). Since pain needs to be located in at least 4 areas according to the 2016 revision, the previous criterion for diagnosis, WPI of 3-6 and SS scale score ≥9 was changed to WPI of 4-6 and SS scale score ≥9.

The 2010 and 2011 ACR preliminary diagnostic criteria are extremely similar. Their difference is that the 2010 criteria are physician-based and can be used in clinical practice for the diagnosis of fibromyalgia, while the 2011 criteria are self-reported and can be used only in survey research. According to the 2010 criteria the SS scale assesses a wide range of somatic symptoms, which makes them impractical for use in questionnaires. With the 2016 revision the assessment of somatic symptoms that is included in the SS scale is limited to headaches, pain and cramps in the lower abdomen and depression. In this way, there is no longer need for different criteria for clinical practice and for survey research. The same criteria can be used in both settings having 2 different methods of administration.

One prerequisite for diagnosis of fibromyalgia according to the 2010 ACR preliminary diagnostic criteria is the patient not to have a condition that would otherwise explain the pain. The authors of these criteria clarified that this does not mean that the diagnosis of fibromyalgia is an exclusion diagnosis. However, this phrasing was not considered clear enough and caused significant misunderstanding. In this way this criterion was removed in the 2016 revision. The diagnosis of fibromyalgia can be valid even if there is another condition that can cause the pain that is attributed to fibromyalgia. According to this definition fibromyalgia can coexist with other clinically significant conditions that can cause pain.

Table 4. 2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria
Criteria: A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met: 1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or Widespread pain index (WPI) 4-6 and symptom severity (SS) scale score ≥9. 2) Generalized pain: Pain must be present in at least 4 of 5 regions. Jaw, chest, and abdominal pain are not included in generalized pain definition. 3) Symptoms have been generally for at least 3 months. 4) A diagnosis of fibromyalgia is valid irrespective of other diagnoses. A diagnosis of fibromyalgia does not exclude the presence of other clinically important illnesses. Ascertainment: 1) WPI Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain? (Score will be between 0 and 19)
	Region 1: Left Upper Region -Jaw, left * -Shoulder girdle, left -Upper arm, left -Lower arm, left	Region 2: Right Upper Region -Jaw, right * -Shoulder girdle, right -Upper arm, right -Lower arm, right	Region 5: Axial Region -Neck -Upper back -Lower back -Chest * -Abdomen *
	Region 3: Left Lower Region -Hip (buttock, trochanter), left -Upper leg, left -Lower leg, left	Region 4: Right Lower Region -Hip (buttock, trochanter), right -Upper leg, right -Lower leg, right
	* Not included in generalized pain definition
2) SS scale score The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the sum of the number of 3 symptoms (headaches, pain or cramps in lower abdomen, depression) (The final score is between 0 and 12) - For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale: 0 = no problem 1 = slight or mild problems, generally mild or intermittent 2 = moderate, considerable problems, often present and/or at a moderate level 3 = severe: pervasive, continuous, life-disturbing problems Fatigue (0-3) Waking unrefreshed (0-3) Cognitive symptoms (0-3) - During the previous 6 months indicate the number of the following symptoms the patient has been bothered by: - Headaches (0-1) - Pain or cramps in lower abdomen (0-1) - Depression (0-1) The fibromyalgia severity (FS) scale is the sum of the WPI and the SS scale

Table 4. 2016 Revisions to the 2010/2011 Fibromyalgia Diagnostic Criteria

Criteria:

A patient satisfies diagnostic criteria for fibromyalgia if the following 3 conditions are met:

1) Widespread pain index (WPI) ≥7 and symptom severity (SS) scale score ≥5 or

Widespread pain index (WPI) 4-6 and symptom severity (SS) scale score ≥9.

2) Generalized pain: Pain must be present in at least 4 of 5 regions.

Jaw, chest, and abdominal pain are not included in generalized pain definition.

3) Symptoms have been generally for at least 3 months.

4) A diagnosis of fibromyalgia is valid irrespective of other diagnoses. A diagnosis of fibromyalgia does not exclude the presence of other clinically important illnesses.

Ascertainment:

1) WPI

Note the number of areas in which the patient has had pain over the last week. In how many areas has the patient had pain?

(Score will be between 0 and 19)

Region 1: Left Upper Region

-Jaw, left *

-Shoulder girdle, left

-Upper arm, left

-Lower arm, left

Region 2: Right Upper Region

-Jaw, right *

-Shoulder girdle, right

-Upper arm, right

-Lower arm, right

Region 5: Axial Region

-Neck

-Upper back

-Lower back

-Chest *

-Abdomen *

Region 3: Left Lower Region

-Hip (buttock, trochanter), left

-Upper leg, left

-Lower leg, left

Region 4: Right Lower Region

-Hip (buttock, trochanter), right

-Upper leg, right

-Lower leg, right

* Not included in generalized pain definition

2) SS scale score

The SS scale score is the sum of the severity of the 3 symptoms (fatigue, waking unrefreshed, cognitive symptoms) plus the sum of the number of 3 symptoms (headaches, pain or cramps in lower abdomen, depression)

(The final score is between 0 and 12)

- For the each of the 3 symptoms below, indicate the level of severity over the past week using the following scale:

0 = no problem

1 = slight or mild problems, generally mild or intermittent

2 = moderate, considerable problems, often present and/or at a moderate level

3 = severe: pervasive, continuous, life-disturbing problems

Fatigue (0-3)

Waking unrefreshed (0-3)

Cognitive symptoms (0-3)

- During the previous 6 months indicate the number of the following symptoms the patient has been bothered by:

- Headaches (0-1)

- Pain or cramps in lower abdomen (0-1)

- Depression (0-1)

The fibromyalgia severity (FS) scale is the sum of the WPI and the SS scale

AAPT Diagnostic Criteria

In an attempt to improve the recognition of fibromyalgia in clinical practice, the AAPT fibromyalgia working group proposed new diagnostic criteria in 2018 (62). These criteria are similar to the ACR criteria as they require the pain to be generalized (multisite), require the presence of non-pain symptoms and require the symptoms to be present for at least 3 months (Table 5). These diagnostic criteria are more simple than the ACR criteria and they can be easily implemented in primary clinical practice, but some of their aspects have been criticized (63). According to the AAPT criteria the head, the abdomen and the chest are included in the areas that are assessed for the presence of generalized musculoskeletal pain. However, these regions are problematic since pain originating from the teeth, the heart and the bowel can be referred to these areas. Additionally, the AAPT criteria do not have the ability to quantify the severity of fibromyalgia as, apart from the generalized pain, they only assess the presence or absence of the 2 most common non-pain symptoms of fibromyalgia, abolishing all other somatic symptoms. Compared with the 2016 ACR diagnostic criteria, individuals with less symptom severity and fewer pain sites can be classified as fibromyalgia patients, when the AAPT diagnostic criteria are used (64).

Table 5. AAPT Diagnostic Criteria
Criteria: 1) Multisite pain defined as 6 or more pain sites from a total of 9 possible sites: - Head - Left arm - Right arm - Chest - Abdomen - Upper back and spine - Lower back and spine, including buttocks - Left leg - Right leg 2) Moderate to severe sleep problems or fatigue 3) Multisite pain plus fatigue or sleep problems must have been present for at least 3 months NOTE. The presence of another pain disorder or related symptoms does not rule out a diagnosis of fibromyalgia. However, a clinical assessment is recommended to evaluate for any condition that could fully account for the patient’s symptoms or contribute to the severity of the symptoms.

Table 5. AAPT Diagnostic Criteria

Criteria:

1) Multisite pain defined as 6 or more pain sites from a total of 9 possible sites:

- Head

- Left arm

- Right arm

- Chest

- Abdomen

- Upper back and spine

- Lower back and spine, including buttocks

- Left leg

- Right leg

2) Moderate to severe sleep problems or fatigue

3) Multisite pain plus fatigue or sleep problems must have been present for at least 3 months

NOTE. The presence of another pain disorder or related symptoms does not rule out a diagnosis of fibromyalgia. However, a clinical assessment is recommended to evaluate for any condition that could fully account for the patient’s symptoms or contribute to the severity of the symptoms.

EPIDEMIOLOGY

The prevalence of fibromyalgia depends on the criteria used to define it. Most studies use either the 1990 ACR classification criteria or the modified 2010 ACR preliminary diagnostic criteria (2011 ACR survey criteria) and the prevalence varies between 2-4% (65). Using the 2016 ACR diagnostic criteria the prevalence of fibromyalgia in the general population is 3-4% while with the AAPT diagnostic criteria the prevalence of fibromyalgia is 73% higher, ranging from 5% to 7% (64). In the general population the prevalence increases with age from 2% at the age of 20 to 8% at age of 70. Fibromyalgia appears more often in relatives of patients suffering from fibromyalgia (66), whereas there is a significant difference on the women to men ratio depending on the criteria used to define fibromyalgia. When the 1990 ACR classification criteria are used the women to men ratio is 7:1 while when the 2011 ACR survey criteria are used, that do not use the tender point count, the ratio ranges from 4:1 to 1:1 (58,67–69). Using the 2016 ACR or the AAPT diagnostic criteria there is no statistically significant difference in the prevalence of fibromyalgia between women and men (64).

DIFFERENTIAL DIAGNOSIS

Several conditions can mimic or overlap with fibromyalgia. In order to reach a differential diagnosis, careful history taking should be followed by a thorough physical examination. Careful neurologic and musculoskeletal examination needs to be performed in all fibromyalgia patients in order to exclude the presence of such conditions. Mood and functional impairment should also be evaluated. This can be easily performed using simple self-administered questionnaires. Patients with obvious mood disturbances should have a formal evaluation by a mental health professional. Baseline blood tests should be limited to a complete blood count, erythrocyte sedimentation rate, a comprehensive metabolic panel, and thyroid function tests. These tests are usually normal in fibromyalgia patients. Consequently, the identification of abnormalities in any of these examinations might suggest that a different condition is present. Additional tests are not recommended for a diagnosis, unless they are clinically indicated. The disorders that can mimic and/or overlap with fibromyalgia and the characteristic clinical features which differentiate them from fibromyalgia are described in Table 6. The clinical features of fibromyalgia, chronic fatigue syndrome (systemic exertion intolerance disease), depression, migraine, and irritable bowel syndrome often overlap being so interchangeable that some authors consider that these conditions should be approached as a “spectrum” of associated disorders (10). They are also considered as part of the spectrum of post-traumatic stress disorder (70,71).

Table 6. Disorders that can Mimic and/or Overlap with Fibromyalgia Along with Characteristic Clinical Features that Differentiate Them from Fibromyalgia
Disorders	Differentiating clinical features
Rheumatoid arthritis, Systematic Lupus Erythematosus, Sjögren’s syndrome	· Characteristic synovitis and systemic features of connective tissue disease, apart from musculoskeletal pain, fatigue, Raynaud phenomenon, dry eyes and dry mouth, are usually not features of fibromyalgia. · Routine serologic tests are not recommended because of low positive predictive value.
Ankylosing spondylitis, other inflammatory back conditions	· Generally, there is normal spinal motion in fibromyalgia. · Characteristic radiologic features of these disorders are not present in fibromyalgia.
Polymyalgia rheumatica	· Tender points are not always present in polymyalgia rheumatica. · Stiffness is more prominent than pain in polymyalgia rheumatica. · Most patients with polymyalgia rheumatica have increased erythrocyte sedimentation rate, while it is normal in fibromyalgia. · Patients with polymyalgia rheumatica respond extremely well to modest doses of corticosteroids, in contrast to fibromyalgia patients.
Inflammatory myositis, metabolic myopathies	· Myositis and myopathies can cause muscle weakness and muscle fatigue, but they are not usually associated with diffuse pain. · Patients with myositis or myopathies have abnormal muscle enzyme tests and specific histopathologic findings on muscle biopsy, in contrast to fibromyalgia patients (muscle biopsy should be limited to cases that there is clinical evidence suggestive of myositis or myopathy).
Statin myopathy	· Statin myopathy symptoms are limited to muscle weakness and pain without other symptoms associated with fibromyalgia. · Statin myopathy pain is temporally associated with statin therapy. · Statin myopathy can be associated with abnormal muscle enzyme tests.
Infection: chronic viral infection (e.g., infectious mononucleosis, HIV, HTLV, HBV, HCV, Lyme disease), long COVID	· In fibromyalgia patients there is no objective evidence of inflammation or organ system dysfunction
Hypothyroidism	· Although thyroid autoantibodies are common in fibromyalgia patients, thyroid function tests are usually normal.
Hyperparathyroidism	· Hypercalcemia is not present in fibromyalgia.
Cushing’s syndrome	· Cushing’s syndrome is associated with muscle weakness rather than pain. · The characteristic facial and skin signs of Cushing’s syndrome are not present in fibromyalgia.
Adrenal insufficiency	· Adrenal insufficiency causes severe exhaustion, while it is not typically associated with chronic widespread pain.
Hypophosphatasia	· Most hypophosphatasia patients have low alkaline phosphatase
Neurologic diseases: peripheral neuropathies, cervical radiculopathy, entrapment syndromes (e.g., carpal tunnel syndrome), multiple sclerosis, myasthenia gravis	· Multiple sclerosis and myasthenia gravis are associated with post-exercise muscle and generalized fatigue, but not with widespread pain. · Thorough neurologic examination can reveal neurologic signs characteristic of specific diseases.
Myofascial pain syndromes (they may include other common regional pain disorders such as tension headaches, occupational overuse syndrome, cumulative trauma disorder, work related musculoskeletal disorder,idiopathic low back and cervical strain disorders, chronic pelvic pain temporomandibular disorderand interstitial cystitis)	· In myofascial pain syndromes the pain and the tenderness is confined in one anatomic region
Chronic fatigue syndrome (systemic exertion intolerance disease)	· Criteria for the diagnosis of chronic fatigue syndrome: 1. According to the modified United States Centers for Disease Control and Prevention chronic fatigue syndrome is diagnosed when two criteria are fulfilled (72): i) Clinically evaluated, unexplained, persistent or relapsing fatigue that is of new or definite onset; is not the result of ongoing exertion; is not alleviated by rest; and results in substantial reduction in previous levels of occupational, educational, social, or personal activities ii) Four or more of the following symptoms that last six months or longer: - Impaired memory or concentration - Post-exertional malaise where physical or mental exertions bring on "extreme, prolonged exhaustion and sickness" - Unrefreshing sleep - Muscle pain - Arthralgia in multiple joints - Headaches of new kind or greater severity - Frequent or recurring sore throat - Tender cervical or axillary lymph nodes 2. According to the proposed diagnostic criteria of the United States Institute of Medicine the chronic fatigue syndrome (systemic exertion intolerance disease) is diagnosed when two criteria are fulfilled (73): i) All of the following symptoms: - A substantial reduction or impairment in the ability to engage in pre-illness levels of occupational, educational, social, or personal activities that persists for more than 6 months and is accompanied by fatigue, which is often profound, is of new or definite onset (not lifelong), is not the result of ongoing excessive exertion, and is not substantially alleviated by rest - Post-exertional malaise* - Unrefreshing sleep* ii) Two or more of the following manifestations: - Cognitive impairment* - Orthostatic intolerance - * The diagnosis should be questioned if patients do not have these symptoms at least half of the time with moderate, substantial, or severe intensity. · Chronic widespread pain is not included in the criteria for diagnosis of chronic fatigue syndrome
Psychiatric disorders: depression, anxiety disorders, posttraumatic stress disorder	· In fibromyalgia patients with a concurrent psychiatric disorder, the attribution of symptoms to fibromyalgia or the psychiatric disorder is not always possible.
Sleep disorders: obstructive sleep apnea, restless legs syndrome, periodic limb movement disorders	· Detail history can identify the majority of the primary sleep disorders. · Chronic widespread pain is uncommon in primary sleep disorders.
Irritable bowel syndrome	· According to the 2009 American College of Gastroenterology recommendations for the diagnosis of irritable bowel syndrome, it is defined by abdominal pain or discomfort that occurs in association with altered bowel habits over a period of at least three months (74).
Temporomandibular disorders	· Temporomandibular disorders are characterized by recurrent facial/jaw pain and/or limitation in jaw opening occurring in the past six months.
Tension – Migraine headache	· Tension – migraine headache is characterized by recurrent headaches (at least five for migraine, at least 10 for tension-type) lasting 30 minutes.
Interstitial cystitis	· According to the American Urological Association guidelines interstitial cystitis is defined as an unpleasant sensation (pain, pressure, discomfort) perceived to be related to the urinary bladder, associated with lower urinary tract symptoms of more than six weeks duration, in the absence of infection or other identifiable causes (75).

HIV: human immunodeficiency virus, HTLV: human T-lymphotropic virus, HBV: hepatitis B virus, HCV: hepatitis C virus, COVID: coronavirus disease

PATHOPHYSIOLOGICAL MECHANISMS

Pain sensitivity in the population occurs over a wide continuum, forming a classic bell-shaped curve, just like any other physiologic variable. Genetic predisposition in combination with environmental factors, determine the place that each individual takes in this continuum. People who are placed in the right end of this curve are very sensitive to pain and they can probably develop pain even without having any inflammation or damage in the peripheral tissues. This pain can be either regional or widespread (39).

In the past fibromyalgia was thought to be a primary muscle disease. However, controlled studies found no evidence of significant pathologic or biochemical muscle abnormalities that can be the cause of chronic widespread pain and tenderness. Most investigators believe that any muscle pathology is secondary to chronic pain and inactivity, rather than primary in nature (76–79). Current research suggests that altered central nervous system (CNS) physiology might underlie the symptoms of fibromyalgia. Abnormal central sensory processing of pain signals seems to play a significant role in the pathogenesis of fibromyalgia. This dysregulation of the nociceptive system can arise from a combination of interactions between neurotransmitters, cytokines, hormones, the autonomic nervous system, behavioral constructs, and external stressors.

Abnormalities in Sensory Processing

Fibromyalgia overlaps with several other similar syndromes including chronic fatigue syndrome (systemic exertion intolerance disease) and myophasial pain syndrome (Table 6). It has been proposed that these disorders should be considered as members of the central sensitivity syndromes (Table 7). These similar and overlapping syndromes are bound by the common mechanism of central sensitization that involves hyper-excitement of the second-order neurons, especially the wide-dynamic-range neurons (WDR) in the dorsal horns of the spinal cord, by various synaptic and neurotransmitter/neuromodulator activities (6). Central sensitization is clinically and physiologically characterized by hyperalgesia, allodynia, expansion of the receptive field (pain expanding beyond the area of the peripheral nerve supply, after the application of a nociceptive stimulus), prolonged electrophysiological discharge, and an after-stimulus unpleasant quality of the pain (e.g., burning, throbbing, tingling or numbness). Parallel to central sensitization, temporal summation takes place in the second-order neurons. It is characterized by a progressive increase in electrical discharges (and consequently increase in the perceived intensity of pain) in response to repetitive short noxious stimuli. Temporal summation involves the production of second pain, which is described as dull or burning, and leaves an after-stimulus unpleasant sensation (80).

Table 7. Central Sensitivity Syndromes
· Fibromyalgia · Chronic fatigue syndrome (systemic exertion intolerance disease) · Irritable bowel syndrome · Tension type headaches · Migraine · Temporomandibular disorder · Myophasial pain syndrome · Restless leg syndrome · Periodic limb movements in sleep · Primary dysmenorrhea · Interstitial cystitis · Posttraumatic stress disorder

Table 7. Central Sensitivity Syndromes

· Fibromyalgia

· Chronic fatigue syndrome (systemic exertion intolerance disease)

· Irritable bowel syndrome

· Tension type headaches

· Migraine

· Temporomandibular disorder

· Myophasial pain syndrome

· Restless leg syndrome

· Periodic limb movements in sleep

· Primary dysmenorrhea

· Interstitial cystitis

· Posttraumatic stress disorder

N-methyl-D-aspartate (NMDA) receptors are mostly responsible for escalation of hyperexcitability of the second-order nociceptive neurons. The role of the major neurotransmitters of the nociceptive system that participate in signal conduction at the level of the spinal cord is briefly illustrated in Figure 3 (6).

Figure 3. The role of the major neurotransmitters of the nociceptive system that participate in signal conduction at the level of the spinal cord. SP: Substance P, NGF: nerve growth factor, NMDA: N-methyl-D-aspartate, D: dopamine.

The second-order neurons have ascending projections to the thalamus, hypothalamus, the limbic system and the somatosensory cortex. These supraspinal structures are involved in the sensory, evaluative and affective dimensions of pain (e.g., unpleasantness, emotional reaction). Several descending pathways from the cortico-reticular system, locus ceruleus, hypothalamus, brain stem, and local spinal cord interneurons terminate to the dorsal horn cells. These pathways utilize neurotransmitters that include serotonin (5-HT), norepinephrine, γ-amino-butyric acid (GABA), enkephalins and adenosine (6). This descending system, once thought to be predominantly inhibitory, is now known to have a facilitatory potential (81). Evidence suggests that the 5-HT₃ receptor has a facilitatory function, while the 5H-T_1Areceptor is inhibitory. The ascending and descending pathways should not be considered as dichotomous in function. They are interactive and their functions are bidirectional. Both pathways can either facilitate or inhibit pain, depending on the site of action and the neurotransmitters that are used (6).

The dysregulation of the nociceptive system, either at the level of the dorsal horns of the spinal cord, or at the level of the ascending and descending pathways, can lead to its hyper-excitability. In other words, it can lead to central sensitization. Several factors may amplify and sustain central sensitization through interactive and synergistic actions. These factors are summarized in Table 8 (80). Central sensitization can become self-sustained, even when the event that triggered it no longer exists, due to long-term CNS plasticity.

Table 8. Factors that may Amplify and Sustain Central Sensitization
· Genetics · Sympathetic over-activity · Endocrine dysfunctions · Viral infection · Peripheral nociception generators (e.g., arthritis) · Poor sleep · Environmental stimuli (e.g., weather, noise, chemicals) · Psychological distress (e.g., adverse childhood experience)

Table 8. Factors that may Amplify and Sustain Central Sensitization

· Genetics

· Sympathetic over-activity

· Endocrine dysfunctions

· Viral infection

· Peripheral nociception generators (e.g., arthritis)

· Poor sleep

· Environmental stimuli (e.g., weather, noise, chemicals)

· Psychological distress (e.g., adverse childhood experience)

Neuroimaging studies provide moderate evidence for structural changes in the brain of patients with fibromyalgia. Gray matter volume appears to be reduced in areas related with pain processing, such as the cingulate, the insular, and the prefrontal cortices (82). Functional MRI studies reveal alterations in the functional connectivity of brain areas responsible for pain processing and provide support of functional dysregulation of the ascending and descending pain pathways in fibromyalgia patients (82–85). Additionally, alterations in neuronal activity between the ventral and the dorsal spinal cord have been demonstrated in fibromyalgia patients (86).

Although nearly all of the research on sensory processing in fibromyalgia has focused on the processing of pain, there are some data suggesting a more generalized disturbance in sensory processing. There is evidence that fibromyalgia patients have a hypersensitivity to unpleasant stimuli of other sensory systems. For example, many patients experience reduced tolerance to loud noises, bright lights, odors, drugs, and chemicals (87,88).

Neurotransmitters

The levels of Substance P (SP) in the cerebrospinal fluid (CSF) in patients with fibromyalgia are significantly increased compared to normal individuals, whereas CSF levels of serotonin metabolites are decreased, as are metabolites of dopamine and norepinephrine (89).

The first direct evidence that fibromyalgia patients may have abnormal dopamine response to pain originated from positron emission tomography (PET) competitive binding studies using the D₂/D₃ receptor antagonist [¹¹C] raclopride. It was shown that dopamine is released in response to tonic toxic noxious muscle stimulation, but not after non-painful muscle stimulation in healthy human subjects. In contrast the dopamine response in fibromyalgia patients did not differ between painful and non-painful muscle stimulation (87). There are indications that disturbances of the opioidergic system occur in fibromyalgia patients, as there is an up-regulation of opioid receptors in the periphery, with a reduction of the brain opioid receptors (90,91). This implies an increased baseline endogenous opioidergic activity. Opioids can activate glial cells, via a non-stereoselective activation of toll-like receptor 4 (TLR4). Glial cells in turn can mediate pain by releasing neuroexcitatory, pro-inflammatory products (92).

In a study where fibromyalgia patients were evaluated for cortical excitability and intracortical modulation using transcranial magnetic stimulation of the motor cortex, it was shown that there were deficits in intracortical modulation of GABAergic and glutamatergic mechanisms (93). Diminished inhibitory neurotransmission resulting from lower concentrations of GABA within the right anterior insula of patients with fibromyalgia was documented using proton magnetic resonance spectroscopy (94). Evidence for enhanced glutaminergic neurotransmission in fibromyalgia patients is derived from studies that used magnetic resonance spectroscopy. It was shown that fibromyalgia patients have significantly higher levels of glutamine within the posterior insula and in the right amygdala (95,96). The levels of brain-derived neurotrophic factor, which is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system, have been found to be increased both in the brain and in the plasma of fibromyalgia patients (97).

Cytokines

Although fibromyalgia is not considered an inflammatory disorder, the interaction of immunological mechanisms with pain physiology, has led to the identification of alterations in the levels of various cytokines (98). However, it is not clear whether cytokine changes are the cause of pain in these patients, or just its consequence.

The serum levels of interleukin 1 receptor antibody (IL-1Ra), IL-6, and IL-8, and the plasma levels of IL-8 are higher in fibromyalgia patients, compared to controls (99). Inflammatory cytokines such as IL-1β, IL-6 and tumor necrosis factor alpha (TNFα) have been detected in skin biopsies taken from fibromyalgia patients, possibly indicating an element of neurogenic inflammation (100). Lower levels of the anti-inflammatory cytokines IL-4 and IL-10 have been reported in fibromyalgia patients compared to healthy controls (101,102). However, the interpretation of cytokine levels is not always easy. Most cytokines are expressed in low levels and a sensitive bioassay is needed for their detection. Consequently, a negative result can merely be the ramification of a not sensitive enough method. Additionally, cytokine levels can be vigorously influenced by a number of factors, including circadian rhythmicity, physical activity, and co-morbid conditions, including depression.

Inflammatory cytokines like IL-1β, IL-6 and TNFα can elicit pain, induce hyperalgesia and they are associated with neuropathic pain (103), although they do not appear to be involved in “normal” pain. Although serum cytokines do pass the blood brain barrier the release of pro-inflammatory cytokines by immune cells in the body leads, in turn, to release of pro-inflammatory cytokines by glial cells within the brain and spinal cord (104).Inflammatory cytokines like IL-1β, IL-6 and TNFα can also cause activation of the hypothalamic-pituitary-adrenal (HPA) axis alone, or in synergy with each other. There is evidence to suggest that IL-6, which is the main endocrine cytokine, plays the most significant role in the immune stimulation of the axis, especially in chronic inflammatory stress (105). IL-6 can stimulate the hypothalamic secretion of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), leading to the increase of serum adrenocorticotropic hormone (ACTH) and cortisol levels (106).

Hypothalamic-Pituitary-Adrenal Axis

There is substantial data supporting an abnormal function of the HPA axis in fibromyalgia. However, the results from studies on the HPA axis function in fibromyalgia patients are relatively heterogeneous and partially contradictory (107). The 24-hour urinary free cortisol (UFC) has been found to be reduced or normal (108). Findings regarding alterations in diurnal variation of cortisol secretion are also inconsistent. Although normal diurnal patterns of ACTH and cortisol have been reported, there is data demonstrating flattened cortisol diurnal rhythm with normal morning peak and higher evening cortisol, levels (109). It has been demonstrated that there is a significant decrease in the rate of decline from acrophase (peak) to nadir for diurnal cortisol levels in fibromyalgia patients, compared to controls, while there is no change in the ACTH to cortisol ratio (108). This implies a decreased ability of the HPA axis to return to baseline after a physiologic stimulation by meals, several other activities or even pain (108). Decreased morning cortisol release and reduced frequency of cortisol pulses over 24 hours have also been reported. There is evidence to suggest that the reduced cortisol release in fibromyalgia patients is associated with depressive symptoms and experiences of childhood trauma (109).

In line with studies suggesting reduced adrenal output in fibromyalgia patients, reduced cortisol secretion has been reported in response to pharmacological challenge with synthetic ACTH_1-24 and insulin tolerance test (109). Patients with fibromyalgia exhibit increased ACTH, but normal cortisol response to CRH stimulation, compared to controls. This finding suggests a sensitization of the pituitary in combination with a degree of adrenal insufficiency (109). Arginine vasopressin (AVP), an ACTH secretagogue, has been found to be more increased in response to the postural challenge test in fibromyalgia patients, compared to controls (110). Alterations in the feedback regulation of HPA axis have also been reported in fibromyalgia patients, using the overnight dexamethasone suppression test (DST). Increased rates of non-suppression following the standard (1 mg) DST have been reported in fibromyalgia patients, compared to controls, but this finding was difficult to be interpreted as it was associated with depression. Interestingly, other studies have revealed lower rates of non-suppression in fibromyalgia patients (111).

There are indications that there is a dissociation between total and free cortisol levels in fibromyalgia patients, with normal salivary and plasma free cortisol despite diminished total cortisol levels. One possible explanation of this finding is a reduced concentration of the glucocorticoid binding globulin (CBG). Reduced levels of CBG have been reported in fibromyalgia patients compared to controls. It is of particular interest that chronic social stress might result in reduced CBG levels, whereas IL-6 and IL-1β that can also inhibit the production of CBG may contribute further (109). Apart from HPA axis abnormalities in fibromyalgia patients, abnormal levels of growth hormone have also been found in some, but not all reports (112). On the other hand the levels of sex hormones have not been clearly shown to differ between female fibromyalgia patients and controls (113).

Autonomic Nervous System

Sympathetic hyperactivity, often associated with sympathetic hypo-activity in response to stressors, or parasympathetic underactivity has been described in fibromyalgia. Attenuated sympathetic and parasympathetic activity was demonstrated in a study where fibromyalgia patients and healthy controls were assessed for 24 hours in a controlled hospital setting, including relaxation, a test with prolonged mental stress and sleep. The urinary catecholamine levels were found to be lower in fibromyalgia patients compared to controls. Patients with fibromyalgia had significantly lower adrenaline levels during the night and the second day of the study, and significantly lower dopamine levels during the first day, the night, and the second day. Furthermore, heart rate during relaxation and sleep was significantly higher in patients than in controls (114). In another study, plasma catecholamines, ACTH, and cortisol levels were reduced in 16 fibromyalgia patients compared to 16 healthy controls while performing static knee extension until exhaustion (115). Nocturnal heart rate variability indices have been shown to be significantly different in fibromyalgia women compared to healthy individuals, indicating a sympathetic predominance (116). In addition, orthostatic hypotension and increased pain in response to tilt table test have been described along with increased resting supine heart rate and decreased heart rate variability (117,118). IL-6 administration causes exaggerated norepinephrine responses and increases in heart rate, as well as delayed ACTH release, suggesting an incapacitated stress-regulating system (119). In-vitro testing of beta adrenergic receptor mediated cyclic AMP generation has revealed decreased responsiveness to beta-adrenergic stimulation (120).

Overall, it has been suggested that sympathetic dysfunction can not only cause diffuse pain, but also contribute to other symptoms like sleep disturbances, due to sustained nocturnal sympathetic activity, and fatigue, due to deranged sympathetic response to stress (6).

Psychological, Cognitive, and Behavioral Factors

Pain apart from a sensory-discriminative dimension, which includes the location and the intensity of pain, has a very significant psychological component. This includes the affective dimension of pain, the emotional valance of pain in other words, as well as attention and cognitive aspects, which are based on CNS mechanisms. Emotion and selective attention can enhance pain perception, with the involvement of the descending pathways that have a facilitatory effect on the spinal cord dorsal horn neurons (80). Catastrophizing has been shown to be related to decreased pain threshold and tolerance to heat stimuli in fibromyalgia patients. However there is a subgroup of fibromyalgia patients that is very tender, despite the fact that they do not catastrophize and they have a moderate control over their pain (11). A fMRI study has shown that although depression is associated with the magnitude of neuronal activation in brain regions that process the affective-motivational dimension of pain, neither the extent of depression nor the presence of comorbid major depression modulated the sensory-discriminative aspects of pain processing in fibromyalgia patients (121). Catastrophizing, has been associated with increased activity in brain areas related to anticipation, attention and the emotional aspects of pain, as shown by fMRI in response to pressure stimuli. This study also revealed an association between catastrophizing and increased activity in the secondary somatosensory cortex, indicating that the way patients think about their pain might actually influence its sensory processing (80).

Genetic Predisposition

It is currently well established that familial aggregation is a characteristic of fibromyalgia. First degree relatives of fibromyalgia patients are 8.5 times more likely to have fibromyalgia than relatives of patients with rheumatoid arthritis (12). As with other complex and multifactorial syndromes, the occurrence of familial aggregation in the case of fibromyalgia does not necessarily imply a genetic basis. Shared environmental factors and learned patterns of behavior that may evolve within families are equally valid targets of investigation.

Genome-wide association studies have shown significant differences in allele frequencies between fibromyalgia patients and controls. However many of the results are inconsistent, without being replicated. Additionally the small sample size of these studies limits the genetic variants that can be identified only to those with large effects (122–126). The coexistence of other comorbidities in fibromyalgia patients further obscures these results. In a large scale genome-wide association study of 26,749 individuals the overall estimated heritability of fibromyalgia was 14%. There was a significant difference between age groups, with the heritability in individuals less than 50 years of age to be 23.5%, while in those over 60 years of age it was only 7.5% (126).

Although no specific candidate gene has been identified, the following genes have been associated with fibromyalgia:

SEROTONIN TRANSPORTER (5-HTT) GENE

An increased frequency of the S/S genotype of the 5-HTT gene has been found in fibromyalgia patients compared to controls (127,128). However this putative association may be limited to patients with concomitant affective disorders, since it was not confirmed in fibromyalgia patients without depression or anxiety (129).

D₄ RECEPTOR GENE

Polymorphisms affecting the number of repeats in the third cytoplasmic loop of the dopamine D₄ receptor gene have been shown to be significantly decreased in frequency in fibromyalgia patients (130).

CATECHOL-O-METHYL TRANSFERASE (COMT) GENE

The homozygous low activity (met/met) and the heterozygous low activity (val/met) COMT genotypes occur more often in fibromyalgia patients than in controls, whereas the homozygous high activity (val/val) genotype is less frequent (131). However in a meta-analysis COMT gene val(158)met polymorphism was not associated with an increased risk for fibromyalgia (132). The met/met genotype has been associated with greater fibromyalgia illness severity across the domains of pain, fatigue, sleep disturbance, and psychological distress, while fibromyalgia patients with the met/met polymorphism experienced a greater decline in exhibiting a positive attitude on days when pain was elevated than did patients with the val/met or val/val genotype (133).

OPIOID RECEPTOR μ 1 GENE (OPRM1)

The 118G allele frequency has been described to be significantly lower in patients with fibromyalgia than in the control group (134).

ADRENERGIC RECEPTOR GENES

The presence fibromyalgia and its symptom severity is associated with various adrenergic receptor gene polymorphisms (135).

Other genes associated with the regulation of nociceptive and analgesic neuronal pathways

Specific variants of trace amine-associated receptor 1 (TAAR1) gene, regulator of G-protein signaling 4 (RGS4) gene, cannabinoid receptor 1 (CNR1) gene, and glutamate receptor, ionotrophic, AMPA 4 (GRIA4) gene, have been associated with fibromyalgia (123).

External Stressors

Almost all diseases are caused by a combination of genetic predisposition and the effect of environmental factors. We are now beginning to better understand the environmental factors that seem to be important in triggering fibromyalgia. Most of them act as “stressors” that when superimposed onto a deranged stress-response system can lead to the dysregulation of the nociceptive system.

PERIPHERAL PAIN SYNDROME

Pain due to damage or inflammation of peripheral tissues may trigger fibromyalgia. Additionally, small fiber neuropathy can be associated with fibromyalgia (136–138). Chronic localized – regional pain can lead to central sensitization and pain dis-inhibition, causing pain hypersensitivity and widespread pain. Systematic autoimmune diseases can be associated with fibromyalgia too. Approximately 20-25% of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis, have co-morbid fibromyalgia (18). In such cases, it is important to realize that many symptoms may be attributed to fibromyalgia rather than the underlying disorder. This recognition has significant clinical implications.

INFECTIONS

Various infections have been linked to the development of fibromyalgia and chronic fatigue syndrome (systemic exertion intolerance disease). Epstein-Barr virus, parvovirus, Lyme disease, Q fever, HIV and hepatitis C virus (HCV), have been suggested as triggers of fibromyalgia or chronic fatigue syndrome (systemic exertion intolerance disease), but more robust evidence is needed. The role of vaccination in precipitating fibromyalgia and related syndromes is still not clear (139,140).

PHYSICAL TRAUMA

Various forms of physical trauma have been considered as culprits of triggering the pathogenesis of fibromyalgia. Many patients report the initiation or the exacerbation of their symptoms after a traumatic event such as whiplash injury, while increased rates of fibromyalgia have been demonstrated among patients undergoing cervical trauma during motor vehicle accidents (141,142).

PSYCHOLOGICAL DISTRESS

It has been considered that psychological factors that give rise to chronic stress may initiate the chain of events that leads to fibromyalgia. The chronic stress can be a result of the accumulation of daily stress events. Emotional stress, catastrophic events such as war, job loss, marital discord and excess family responsibilities such as caring for sick elders, have been implicated as triggers of fibromyalgia (143). However the data that supports the notion that psychological stress and distress directly causes fibromyalgia is rather weak (39).

MANAGEMENT

The treatment of fibromyalgia is challenging because of our limited understanding of its pathogenesis and the poor response of patients to conventional pain treatments. The aim of the therapy is to relieve pain and increase function using a multimodal individualized therapeutic strategy which, in most cases, includes pharmacologic and non-pharmacologic interventions. Current clinical-based evidence supports the use of a multimodal program that includes education, exercise, cognitive-behavioral approaches and medications. The treatment should be individualized based on the symptoms, the comorbidities and the preferences of the patient, who should be encouraged to participate in the decision-making process of selecting the optimal therapies (144,145). Coexisting disorders are common in fibromyalgia patients. Their identification and effective treatment can have beneficial effects on fibromyalgia symptoms. It is also important to assure that adequate adherence to both pharmacological and non-pharmacological treatment is maintained, so as to achieve the optimal benefit from these treatments.

Non-Pharmacological Management

PATIENT EDUCATION

The first step should be the education of the patient. The patients with fibromyalgia need to understand their illness before any treatment modality is used (146). Providing a diagnosis, “labeling” the patient with fibromyalgia, may have beneficial effects. It has been shown that fewer symptoms and an improvement in health status is noted after the patients are informed of their diagnosis (147,148). The physician should clarify that fibromyalgia is a real illness and the symptoms the patient experiences are not imaginary. The role of neurotransmitters and neuromodulators in pain perception, fatigue, abnormal sleep and mood disturbances should be discussed, so as the patient to understand the rationale of the pharmacologic therapy, especially when antidepressant drugs are used. The significance of good sleep hygiene should be reviewed and poor sleep habits should be addressed. Fibromyalgia patients who are overweight or obese should be informed for the adverse effect of increase body mass index to fibromyalgia symptoms and quality of life (149). For these patients weight reduction should be encouraged. The patient also needs to acknowledge that fibromyalgia is a chronic relapsing condition without though being life-threatening nor deforming.

EXERCISE

Another potent non-pharmacological treatment for fibromyalgia is exercise. It has been reported that an exercise program incorporating aerobic, strengthening, and flexibility elements can lead to greater benefits than a relaxation program. Exercise in fibromyalgia patients should have two major components: strengthening to increase soft-tissue length and joint mobility, and aerobic conditioning to increase fitness and function, reduce fibromyalgia symptoms and improve quality of life (144,150–153). Exercise should be of low impact and of sufficient intensity so as to be able to change aerobic capacity (28). Successful interventions include fast walking, biking, swimming, water aerobics, tai chi, and yoga. Land and aquatic training appears to be equally beneficial (154). An improvement in the severity of fibromyalgia symptoms has also been achieved with web-based exercise programs (155). A gradual incremental increase in physical activity should be encouraged as it is common for fibromyalgia patients to initially perceive an aggravation of their pain and fatigue at the beginning of a training program. It has been suggested that in the presence of exercise-induced pain, the intensity and duration of exercise should be reduced, while its frequency should be maintained, so as to avoid any further decrease in exercise tolerance (144). The type and intensity of the exercise program should be individualized and should be based upon patient preference and the presence of any other cardiovascular, pulmonary, or musculoskeletal comorbidities.

COGNITIVE-BEHAVIORAL APPROACHES

One of the goals of the management should be to help patients understand the effect of thoughts, beliefs and expectations on their symptoms. This can help them to abolish the perception of helplessness and the catastrophizing thoughts that can adversely influence their condition. Patients with greater self-efficacy are more likely to have a good response to treatment programs and experience better outcomes. The beneficial effect of cognitive-behavioral therapies in fibromyalgia patients with anxiety and depression disorders is limited to a reduction of negative mood, while the rest of the patients also demonstrate a reduction of pain and fatigue. Psychologically based interventions, have been proven to be useful when they are compared to no treatment or treatment other than aerobic exercise (156). In a 2021 systematic review and meta-analysis there was high quality evidence that cognitive-behavioral therapy can significantly reduce the pain intensity in fibromyalgia patients for 3 months (157). Preliminary data from functional MRI studies suggest that cognitive-behavioral therapies have the ability to restore the alterations in the functional connectivity of brain areas responsible for pain processing observed in fibromyalgia patients (158,159).

COMPLEMENTARY AND ALTERNATIVE APPROACHES

Acupuncture

Acupuncture is the insertion of needles in the human body. There are different styles of acupuncture depending on the location and the depth the needles are inserted. The inserted needles can be stimulated by heat, electrical current (electro-acupuncture), mechanical pressure (acupressure), or laser (laser acupuncture). The most common type of acupuncture involves skin penetration without stimulation (manual acupuncture). Sham or fake acupuncture is a research tool to control the effects of real acupuncture. It can involve skin contact with the needles without actual penetration or needle insertion in areas other than the ones usually targeted.

In a high quality meta-analysis it was demonstrated that the effects of manual acupuncture on pain, sleep quality and global well-being did not differ significantly from the effects of sham acupuncture. On the contrary electro-acupuncture significantly reduced pain, fatigue, and stiffness, while it improved sleep quality and global well-being when compared to sham acupuncture. Additionally, electro-acupuncture significantly improved pain, stiffness, and global well-being when compared to non-acupuncture. The beneficial effects of acupuncture could be observed at 1 month after treatment, but they were not maintained at 6-7 months (160).

Other

The effectiveness of meditative movement therapies (qigong, yoga, tai chi) on sleep and fatigue improvement and of hydrotherapy on pain reduction has been supported by some studies (161,162). A number of other modalities has also been utilized for the treatment of fibromyalgia including biofeedback, chiropractic therapy, massage therapy, hypnotherapy, guided imagery, electrothermal therapy, phototherapeutic therapy, music therapy, journaling / storytelling, static magnet therapy, transcutaneous electrical nerve stimulation, and transcranial direct current stimulation. However there are no well-designed studies to advocate their general use (145).

Pharmacologic Treatment

A wide range of drugs has been used in the treatment of fibromyalgia including antidepressants, sedatives, muscle relaxants and antiepileptic drugs. The choice of medication is influenced by patient preference; prominence of particular symptoms, including fatigue, insomnia, and depression; potential adverse effects; patient tolerance of individual medications; cost and regulatory limitations on prescription choice (163,164). Nonsteroidal anti-inflammatory drugs and opioids, although often prescribed for fibromyalgia, are not an effective form of treatment (39,165). However analgesics and anti-inflammatory medications can be useful in case of coexisting conditions that cause regional pain, like arthritis, which can aggravate or trigger the fibromyalgia symptoms. Regarding opioids, with the exception of tramadol, apart from not being effective for the treatment of fibromyalgia symptoms, their long-term use also curries a dose-dependent risk for serious adverse effects, including overdose, abuse, fractures, myocardial infarction and sexual dysfunction (166). Additionally opioids in fibromyalgia patients can reduce the effectiveness of psychological therapy (167), while their long-term use can cause sleep disturbances (168).

Patients should be informed that for most pharmacologic therapies several weeks may be needed until they experience a benefit. Initially a single drug should be administered. However, in the case of non-responsiveness combination therapy should be considered. Since therapeutic responses are rarely durable, physicians should not be surprised when the initial efficacy of a medication is abolished. Successful treatment of fibromyalgia may require regular reassessment and possible rotation or combination of medications (169). Adequate dose prescription and patient adherence are significant for the effectiveness and tolerability of pharmacologic treatment (170). The doses of the most commonly used medications with strong and moderate evidence of effectiveness are shown in Table 9.

ANTIDEPRESSANTS

Tricyclic Antidepressants (TCAs)

TCAs are often used as initial treatment for fibromyalgia. Their analgesic effect is independent of their antidepressant action and is thought to be mediated by inhibition of norepinephrine (rather than serotonin) reuptake at spinal dorsal horn synapses, with secondary activity at the sodium channels. The most widely studied drugs of this group are amitriptylineand cyclobenzaprine. They should be administered at lower doses than those required for the treatment of depression, a few hours before bedtime, and their dose should be escalated very slowly. A clinically important improvement is observed in 25-45% of patients treated with TCAs compared to 20% in those taking placebo (171–175). However their use is limited by the fact that they are ineffective or intolerable in 60-70% of patients (144), while their efficacy may decrease over time (171,176).

Amitriptyline is more efficient compared to the serotonin-norepinephrine reuptake inhibitors duloxetine and milnacipran in reducing pain, sleep disturbance, and fatigue, without differences in acceptability, as it was shown in a systematic review and meta-analysis (177). In a 2022 network meta-analysis comparing amitriptyline, duloxetine and pregabalin it was shown that treatment with amitriptyline 25 mg was superior to duloxetine and pregabalin for the reduction of pain intensity for at least 50% (178). The combination of 20 mg of fluoxetine in the morning with 25 mg of amitriptyline at bedtime has been shown to be more effective than either medication alone (179). Side effects of amitriptyline include dry mouth, constipation, fluid retention, weight gain, difficulty in concentrating and possibly cardiotoxicity.

Cyclobenzaprine has a similar tricyclic structure and presumed mode of action with amitriptyline in fibromyalgia, but is thought to have minimal antidepressant effect (163). A meta-analysis of five placebo-controlled trials has revealed improvement of the global functioning, with a similar effect size as this reported for amitriptyline. The group that received cyclobenzaprine had a significant decrease in pain for 4 weeks, compared to those treated with placebo, but the decrease in pain was not significantly different after 8 and 12 weeks. Sleep was improved at all time points in both cyclobenzaprine and placebo groups, while no effect was noted on fatigue (171–173). It has been demonstrated that the use of very low-dose cyclobenzaprine (1 to 4 mg at bedtime) can improve the symptoms of fibromyalgia, including pain, fatigue, and depression, compared to symptoms at baseline and to placebo. Significantly more patients who received the very low-dose of cyclobenzaprine experienced improved restorative sleep, based upon analysis of cyclic alternating pattern of sleep by electroencephalography. The increase in nights with improved sleep by this measure correlated with improvements in fatigue and depression (180).

Desipramine has fewer anticholinergic and sedative effects than other TCAs, which can make it a possible alternative, although its efficacy is not well studied in fibromyalgia.

Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs)

SNRIs are similar to TCAs in their ability to inhibit the reuptake of both serotonin and norepinephrine, but they differ from TCAs in being devoid of significant activity at other receptor systems, resulting in diminished side effects and increased tolerance. Venlafaxine, duloxetine and milnacipran have been shown to be effective in diminishing fibromyalgia symptoms (147,181,182). These drugs can be used in fibromyalgia patients who do not respond to a trial of low-dose TCAs or who have intolerable side effects. They can also be administered as an alternative to amitriptyline for initial therapy especially for patients with significant fatigue or depression. Of these medications duloxetine and milnacipranare better studied and they are preferred to be administered to patients with fibromyalgia. There are more limited data regarding the efficacy of venlafaxine for fibromyalgia, while withdrawal symptoms if a dose is missed occur more often, because of the short half-life of this medication (183). A meta-analysis has shown that fibromyalgia patients treated with duloxetine at 60mg daily are more likely to have more than 50% reduction in pain, compared to patients taking placebo (184). However, duloxetine at 30mg daily does not significantly reduce pain (185). The efficacy of duloxetine can be maintained at 3 and 6 months of treatment (186). In a 2018 systematic review and meta-analysis it was shown that duloxetine and milnacipran were not superior to placebo in the frequency of pain relief of at least 50%, but there was a benefit in reducing the pain at least by 30% and in the patient's global impression to be much or very much improved. Additionally, there was not a significant difference in the reduction of fatigue, in the reduction of sleep problems, nor in the improvement of health-related quality of life (187). Another meta-analysis has shown that duloxetine, pregabalin and milnacipran were superior to placebo for pain relief, while duloxetine and pregabalin were superior to milnacipran. These drugs also differed in their effects on sleep disturbances, depression and fatigue (188). A fMRI study in fibromyalgia patients treated with milnacipran and placebo demonstrated that pain reduction with milnacipran treatment was associated with decreased functional connectivity between the insular cortex and the rostral part of the anterior cingulate cortex as well as the periaqueductal gray, while these changes were not demonstrated with the placebo (189).Regarding their side effects, headaches and nausea are more common with duloxetine and milnacipran treatment, while diarrhea is more common with duloxetine treatment. Other side effects related to SNRIs include dry mouth, constipation, somnolence, dizziness and insomnia (188).

Monoamine Oxidase Inhibitors

Monoamine oxidase inhibitors block the catabolism of serotonin, increasing its levels in the brain. It has been indicated that pirlindole and moclobemide have a significant beneficial effect on pain, without a significant effect on sleep nor fatigue. Their use for the treatment of fibromyalgia patients is limited (190).

ANTICONVULSANTS

Antiepileptic medications useful for the treatment of fibromyalgia patients include pregabalin and gabapentin. Both of these medications are structurally related to GABA and they bind with high affinity to the alpha2-delta subunit site of cellular voltage-dependent calcium channels. Although their exact mechanism of action is unknown their therapeutic effects can be mediated by blocking the release of various neurotransmitters. They can be used in cases where other medications initially administered to the fibromyalgia patients become intolerable or ineffective, or as the initial treatment for patients with significant sleep disturbance in addition to pain.

Pregabalin has been reported to be efficient against pain, sleep disturbances and fatigue in fibromyalgia. In a recent meta-analysis a reduction of at least 50% in pain intensity was found in 22-24% of patients taking pregabalin 300-600 mg per day, approximately 9% higher compared to the placebo group. A reduction in pain intensity of at least 30% was found in 39-43% of patients on pregabalin 300-600 mg per day, compared to 28% of patients taking placebo (191). In addition to pain, pregabalin at doses 300-600 mg per day can improve sleep and patient function, as it is demonstrated in a 2018 review of clinical trials, meta-analyses, combination studies and post-hoc analyses (192). The improvement in pain and sleep can be apparent as early as 1-2 days after the onset of treatment (193). In a 2022 network meta-analysis it was shown that treatment with pregabalin 450 mg per day was superior to duloxetine 30 mg for the reduction of pain intensity of at least 30% (178). A randomized placebo-controlled neuroimaging study demonstrated that the reduction in pain intensity from pregabalin was associated with a reduction in connectivity between the posterior insula and the default mode network (DMN) and that pregabalin but not placebo can reduce the response of the DMN to experimental pain (194). It is of interest that baseline patterns of brain connectivity have been used in a machine-learning model to successfully distinguish fibromyalgia patients who have a favourable response to pain intensity after the treatment with milnacipran from those who achieve a reduction of pain intensity after the treatment with pregabalin (195). Common side effects of pregabalin include somnolence, dizziness, weight gain and peripheral oedema. Discontinuation due to side effects is approximately 10% higher in patients treated with pregabalin compared to placebo, while discontinuation due to inefficiency of treatment is 6% lower (191). The intensity of adverse effects and the frequency of discontinuation of the treatment due to adverse effects is dose dependent. It is important for pregabalin to be titrated to the maximally tolerated therapeutic dose for each patient (192).

Gabapentin has been shown to be efficient in treating fibromyalgia associated pain, while it was well tolerated (196). Side effects include dizziness, sedation, lightheadedness, and weight gain. Its efficacy and tolerability is not well studied in fibromyalgia patients, however it can be considered as an acceptable alternative in case pregabalin cannot be administered due to its cost or due to regulatory limitations (197).

SEDATIVE HYPNOTIC AGENTS

Zopiclone and zolpidem have been used in fibromyalgia. It has been suggested that they can improve the sleep and perhaps fatigue, without any significant effects on pain (144).

Sodium oxibate, a precursor of GABA with powerful sedative properties has been shown to improve pain, fatigue and sleep architecture in fibromyalgia (198). However, in view of safety concerns the European Medicines Agency and the US Food and Drug Administration have not approved it for use in fibromyalgia patients.

TRAMADOL

Tramadol has multiple analgesic effects, since it inhibits norepinephrine and serotonin reuptake, and its major metabolite binds weakly to opioid μ receptors (144). The use of tramadol (with or without acetaminophen) is both effective and well tolerated for the management of pain in fibromyalgia (199,200). There are some concerns regarding the long-term potential of abuse of tramadol, although the risk is less than that of more potent narcotic analgesics that have also been used in fibromyalgia.

Table 9. The Doses of the Most Commonly used Medications with Strong and Moderate Evidence of Effectiveness in Fibromyalgia
Drugs	Doses
Tricyclic antidepressants
Amitriptyline	Start 5-10 mg at bedtime, increase up to 25-50 mg
Cyclobenzaprine	Start 10 mg at bedtime, increase up to 30-40mg, decrease to 5mg if 10mg too sedating
Serotonin-norepinephrine reuptake inhibitors
Duloxetine	Start 10-15mg twice daily, gradually increased to 30 mg twice daily
Milnacipran	Start 12.5mg in the morning, gradually increase to 50mg twice daily
Venlafaxine	167 mg per day
Anticonvulsants
Gabapentin	Start 100mg at bedtime, increase to 1200-2400 mg per day
Pregabalin	Start 25-50mg at bedtime, increase to 300-450 mg/day
Other
Tramadol	37.5 mg four times daily

CONCLUSION

Fibromyalgia is a common disease that is often underdiagnosed. Genetic predisposition, in combination with exposure to external stressors may lead to dysregulation of the nociceptive system and to the appearance of clinical symptoms. Fibromyalgia patients do not form a homogenous group with some patients responding adequately to current therapeutic modalities, and some others not experiencing any long-term benefit. Patients treated by primary care physicians in the community have a much better prognosis, compared to patients treated in tertiary referral centers. Certain psychological factors, such as an increased sense of control over pain, a belief that one is not disabled, that pain is not a sign of damage, and behaviors like seeking help from others, decreased guarding during examination, exercising more and having pacing activities are associated with better prognosis. Conversely, catastrophizing is associated with increased awareness of pain and with worsening symptoms.

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Immune System Effects on the Endocrine System

ABSTRACT

Among the most important and complex systems in the human body are the endocrine and immune systems. Emerging research over the last decade has shed light on their remarkable interplay, revealing a multitude of bidirectional communication pathways and reciprocal regulation mechanisms. Endocrine diseases, such as autoimmune thyroiditis, diabetes mellitus type 1 and type 2, osteoporosis, and disorders of the hypothalamic-pituitary-adrenal (HPA) axis, as well as endocrine malignancies, such as thyroid cancer, are highly interconnected with dysregulations of the immune system.Thus, multiple cytokines, chemokines, and evolving inflammatory processes are involved in the pathogenesis of immune-related endocrine disorders, providing potential targets for immune-based therapeutic approaches. In this chapter, we provide a comprehensive overview of the molecular mechanisms underlying these complex endocrine-immune interactions, and discuss the implications of immune system function or dysfunction in endocrine disorders.

INTRODUCTION

The immune system is a host defense system that comprises numerous biological structures and processes to defend the human body against potentially harmful substances and invading pathogens. It functions through a series of coordinated mechanisms, including innate and adaptive immunity. The innate immune response consists of i) phagocytosis by macrophages, neutrophils, monocytes, and dendritic cells, and ii) cytotoxicity by natural killer cells, providing an immediate, nonspecific defense against a wide range of invaders by recognizing common patterns shared by many pathogens (1).

Adaptive immunity, on the other hand, is an acquired, specially designed defense system; it develops over time and utilizes highly specialized immune cells involving antibody-dependent complement or cell-mediated cytotoxicity produced by T cells that recognize injurious agents, such as heat shock proteins or microbial antigens. During adaptive immunity, antigens taken up by antigen-presenting cells (APCs) are presented to T cells through binding with major histocompatibility complex (MHC) molecules on the surface of these cells. Activated CD4 helper T cells stimulate the release of cytokines, such as interleukin (IL)-2, which i) induce T cell proliferation and activation, ii) stimulate killer cell activity by CD8 suppressor T cells, and iii) activate B cells to differentiate into plasma cells and produce antibodies. Naïve T cells differentiate mainly into two main subsets that produce a different set of cytokines and regulate distinct immune functions. T-helper 1 (Th1) cells produce mainly interferon-γ (IFN)-γ, tumor necrosis factor-α (TNF)-, and IL-12 to regulate cell-mediated responses, while T-helper 2 (Th2) cells secrete IL-4, IL-5, and IL-13, to stimulate antibody production. In addition, three other subsets of T helper cells have been identified: Th22, which secrete IL-22, Th17, which secrete IL-17, and Treg, which secrete transforming growth factor (TGF)- b, all of which also play a role in the pathogenesis of autoimmune diseases (2).

Multiple regulatory mechanisms are involved in maintaining central and peripheral T and B cell tolerance (1). Defects in the processes that ensure immune cell tolerance may induce a maladaptive immune response to a self-antigen and lead to the development of autoimmune diseases.

Emerging research of the last few decades has shed light on the remarkable interplay between endocrine and immune systems, revealing a multitude of bidirectional communication pathways and reciprocal regulation.

Autoimmune endocrine diseases, such as Hashimoto thyroiditis, diabetes mellitus type 1 (DM1), and Addison disease, as well as endocrine malignancies, such as differentiated, anaplastic, and medullary thyroid cancer (3), and other endocrine disorders, including diabetes mellitus type 2 (DM2), osteoporosis, as well as a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis response to stress and inflammation, are highly interconnected with dysregulations of the immune system.

This chapter seeks to elucidate the interplay between the endocrine and immune systems, exploring their interconnections and highlighting the impact of their crosstalk on health and disease. We present a comprehensive overview of the molecular mechanisms underlying this interaction and discuss the potential therapeutic implications of targeting the immune system for the management of endocrine diseases.

IMMUNE SYSTEM AND THYROID DISEASE

Autoimmune Thyroid Disease

Autoimmune thyroid disease (AITD) results from a dysregulation of the immune system that leads to loss of tolerance to thyroid antigens and to an autoimmune attack on the thyroid gland. The most common clinical manifestations of AITD are Hashimoto's thyroiditis and Graves’ disease, while less prevalent manifestations are drug-induced thyroiditis, postpartum thyroiditis, or thyroiditis associated with polyglandular syndromes (i.e., autoimmune polyglandular syndromes type 1 and type 2). The underlying molecular mechanisms of AITD involve both circulating autoantibodies and T cell immune mechanisms, while genetic background, as well as cross-reactivity to external antigens (4-6), are also implicated.

There are three major thyroid autoantigens that are targeted during autoimmune thyroid attack and are critical for thyroid homeostasis, namely, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR) (Figure 1).

Figure 1. Thyroid proteins that serve as autoantigens. Thyroglobulin (Tg) function as storage protein in thyroid cells, playing a critical role in the synthesis and release of thyroid hormones. Thyroid peroxidase (TPO) catalyzes iodination of tyrosines in thyroglobulin, which attaches one or two iodine molecules to form monoiodotyrosine (MIT) or diiodotyrosine (DIT), respectively. In addition, thyroid peroxidase catalyzes the coupling of iodotyrosine residues to form triiodothyronine (T3) and thyroxine (T4) attached to thyroglobulin. Thyrotropin receptor (TSHR) is a transmembrane G-protein coupled receptor that upon stimulation by circulating TSH activates the expression of downstream effector genes to regulate thyroid growth, thyrocyte differentiation, and thyroid hormone synthesis. Sodium/iodide symporter (NIS) is a membrane glycoprotein, which actively cotransports two sodium cations per each iodide anion, using the electrochemical sodium gradient generated by the Na+/K+-ATPase. Pendrin is involved in the apical iodide efflux in thyroid cells. It can also exchange chloride and bicarbonate. [Modified by Boguslawska et al (7)].

Thyroglobulin is a soluble glycoprotein homodimer composed of two subunits of ~330 kDa in size and is the most abundant glycoprotein in the thyroid gland. It is the scaffold for the synthesis of thyroid hormones and the storage-form of thyroid hormones inside the gland. Recently, researchers described the first atomic structure of full-length Tg and identified its hormone-forming tyrosine residues (8). Anti-Tg antibodies (TgAb) act mainly through antibody-dependent cytotoxicity cells rather than through complement fixation (7). In AITD, the prevalent TgAb species recognize native rather than denatured antigens and bind to a number of overlapping epitopic domains located mainly in the central region and C-terminal end of Tg. Of note, TgAb in the serum of healthy subjects have a different epitopic pattern (9).

Another major thyroid antigen is TPO, a glycosylated heme-containing homodimer of two 107-kDa transmembrane subunits located in the apical membrane of thyrocytes (Figure 1). It catalyzes the iodination of tyrosyl residues in Tg and the coupling of iodotyrosine residues to form iodothyronines attached to Tg (10). Both humoral and cellular immune responses are directed against TPO. TPO autoantibodies (TPOAb) occur in almost all patients with Hashimoto thyroiditis and approximately 75% of individuals with Graves’ disease (11). In addition, TPOAb may be involved in autoimmune thyroid cell death via antibody-dependent cytotoxic cells and C3 complement-mediated cytotoxicity (7). Specific patterns of TPOAb recognition remain stable in an individual over time and are genetically transmitted through family lineages (12).

Thyrotropin receptor is primarily expressed on the basolateral membrane of thyrocytes (Figure 1) and belongs to the transmembrane G protein-coupled receptor family. The intracellular signaling pathway that is activated by the interaction of TSH with TSHR is indispensable for the synthesis of thyroid hormones and the proliferation of follicular epithelial cells. TSHR-stimulating antibodies (TSAb) act as TSHR agonists and stimulate thyroid growth and production of thyroid hormone in an autonomous and unregulated manner. On the other hand, TSHR-blocking antibodies (TBAb) act as antagonists that block the intracellular signaling of TSH, leading to decrease of thyroid hormone synthesis and subsequently hypothyroidism. Neutral antibodies to TSHR, which may also be detected in the serum of patients with Graves’ disease, bind to the receptor but do not alter its activity (13). The exact antigenic sites of TSHR-specific TBAb and TSAb overlap and are mostly directed to the extracellular A subunit of the receptor (Figure 1) (14,15).

Other thyroid antigens include the iodide transporters, sodium iodide symporter (NIS), and pendrin (Figure 1). The presence of NIS antibodies is increased in AITDs, especially in patients with Graves’ disease, whereas their expression in euthyroid individuals is rare (16). Pendrin is an apical membrane-bound iodide transporter, but the diagnostic value of antibodies against pendrin is rather low (16).

Although the above-described circulating autoantibodies are useful markers of thyroid autoimmunity, it is the T cell immune mechanism, i.e., the loss of immune self-tolerance, that is the core of AITD pathophysiology. Loss of immune self-tolerance may result from either: i) the loss of central tolerance (i.e., disturbed deletion of autoreactive T cells in the thymus), ii) dysfunction of peripheral tolerance (i.e., impaired apoptosis of self-reactive T cells and inhibition of the activity of T-regulatory cells), or iii) disturbed energy (i.e., disturbance of the functional inactivation that prevents the lymphocytes from activating an immune reaction against the antigen).

Patients with AITD express IFN-γ-induced MHC class II molecules, which promotes the presentation of thyroid autoantigens and activates T cells (4). The subsequent infiltration of the thyroid gland by APCs (dendritic cells and macrophages) may be triggered by inflammation resulting from either viral or bacterial infection or exposure to toxins. The common mechanism involved in the initiation and perturbation of the inflammatory processes in AITDs and in other autoimmune endocrine diseases (e.g., type 1 diabetes and Addison disease) is the Th1-cytokine/chemokine axis (17). Upon activation, Th1 lymphocytes produce IFN-γ -and TNF-α, which stimulate thyrocytes and retroorbital cells (in Graves ophthalmopathy) to secrete chemokines (CXCL10, CXCL9, and CXCL11). Chemokines, in turn, bind and activate the CXCR3 receptor on Th1 cells and further enhance IFN-γ and TNF-α secretion in a positive feedback loop which aggravates recruitment and activation of inflammatory cells in the affected organs (Figure 2). In advanced thyroiditis, the thyroid gland is infiltrated by B cells (representing up to 50% of the infiltrating immune cells), as well as cytotoxic T lymphocytes and CD4+ cells (7).

Figure 2. Depiction of the molecular mechanism involved in the inflammatory processes in autoimmune thyroid diseases. Activation of Th1 lymphocytes by antigen presenting cells produce INF-γ -and IL-2 and 12, which stimulate thyrocytes and retroorbital cells to secrete chemokines (CXCL10, CXCL9, and CXCL11). Chemokines bind and activate the CXCR3 receptor on Th1 cells, further enhancing IFN-γ and ILs secretion. APC, antigen presenting cells; IFΝ-γ, interferon gamma; IL-2, interleukin 2; IL-12, interleukin-12; CXCR3, C-X-C Motif Chemokine Receptor 3.

In Hashimoto thyroiditis, the humoral immune response is characterized by the presence of autoantibodies to TPO or Tg and is related to thyroid lymphocytic infiltration. These autoantibodies are themselves cytotoxic or may affect antigen processing or presentation to T cells. Th1 cells are the predominant T cell clones found in patients with Hashimoto thyroiditis: they promote apoptosis of thyrocytes through secretion of IL-12, TNF-α, and INF-γ, which activate cytotoxic T lymphocytes and macrophages (18). In addition, the Toll-like receptor-3 protein is overexpressed in human thyrocytes surrounded by immune cells in all patients with Hashimoto thyroiditis, but not in Graves’ disease or in euthyroid individuals (17).

In patients with Graves’ disease, the predominant antibodies are directed against the TSHR. T cells are activated through the presentation of TSHR peptides and, in turn, trigger B-cells and plasma cells that infiltrate the thyroid to produce autoantibodies directed against the TSHR. Activity of B-cells and plasma cells is also regulated by liver-produced insulin growth factor 1 (IGF1). In contrast to TPO and Tg, TSHR is widely expressed in extrathyroidal tissues and cells, including lymphocytes, adipose tissue, and fibroblasts. In consequence, the presence of TSHR antibodies contributes to the extrathyroidal manifestations of Graves’ disease, such as Graves ophthalmopathy, Graves dermopathy, and Graves-associated thymus hyperplasia (19).

Particularly in Graves ophthalmopathy, the TSHR autoantigen is presented by macrophages and B cells recruited to the orbit (20). Activated T cells, in turn, initiate an immunological attack on the orbital fibroblasts expressing TSHR. In response to the cytokines released by Th cells, orbital fibroblasts and adipocytes —both expressing TSHR— produce and deposit large amounts of glycosaminoglycans (e.g., hyaluronan), leading to increased osmotic pressure and water uptake, swelling of the extraorbital muscles, and increased accumulation of orbital adipose tissue. The process is enhanced by the IGF1/IGF1R signaling pathway in fibroblasts and adipocytes, as well as the crosstalk between TSHR and IGF1R intracellular signaling (21). A similar molecular pathophysiology may underlie Graves dermopathy (19).

NON-CODING RNAs

In recent years, non-coding RNAs, which are known to modulate gene transcription at the post-transcriptional level, have attracted a great deal of attention as potential biomarkers in various endocrine diseases. Although the study of microRNAs and circular RNAs is still in its infancy, these newly discovered non-coding, single-stranded RNA molecules have been implicated in the development and progression of AITDs.

A cluster of biomarkers consisting of miR-205/miR-20a-3p/miR-375/miR-296/miR-451/miR-500a/miR-326 has been reported to be differentially expressed in patients with Hashimoto thyroiditis (22,23). Similarly, multi-miRNA-based biomarkers, such as miR-762/miR-144-3p or miR-210/miR-155/miR-146, were differentially expressed in the serum of patients with Graves’ disease compared to healthy individuals. These dysregulated miRNAs can target key genes involved in the immune response and thyroid function. Regarding their prognostic role, higher miR-21-5p expression is associated with a worse prognosis for patients with Graves’ disease, whereas impaired expression of miR-155 correlates with the size of the goiter (22,24). Further understanding of the role of miRNAs in AITDs could provide valuable insights into disease mechanisms and potentially identify novel therapeutic targets. Data on other non-coding RNAs (such as long-noncoding RNAs and circular RNAs) are scarcer. Impaired expression of n335641, TCONS-00022357-xloc-010919, and n337845 was found in B cells of patients with Graves’ disease (25), while altered expression of 627 circRNAs in PBMCs of patients with Hashimoto thyroiditis has been tested for their potential prognostic value (26).

GUT MICROBIOME

Emerging evidence suggests that the composition and function of the gut microbiome may be involved in AITDs. The gut microbiome refers to the complex community of microorganisms residing in the gastrointestinal tract, which plays a vital role in various aspects of human health, such as prevention of intestinal colonization by pathogenic bacteria, fermentation/degradation of food debris, and production of nutrients. Studies have indicated that alterations in the gut microbiome can influence immune responses and contribute to the development of autoimmune diseases (7). In this context, dysbiosis, or an imbalance in the gut microbiota, has been observed in patients with Hashimoto thyroiditis and Graves’ disease, thus distinguishing them from healthy controls, while it was associated with the stage of disease, the level of thyroid autoantibodies, and the response to therapy.

Targeting the microbiome through dietary interventions or probiotics may represent a promising potential therapeutic avenue. It is therefore of interest to note that in hypothyroid patients treated with LT4, symbiotic supplementation for 8 weeks resulted in decrease of TSH concentration and LT4 dose (27). Moreover, the results of a large clinical study, INDIGO, reported a significant effect of LAB4 probiotics on the gut microbiota composition of patients with Graves’ disease and a temporary reduction in the serum level of IgG and IgA antibodies (28). Further research involving patients from different populations is required to fully understand the relationship between the gut microbiome and AITDs and to explore potential strategies for intervention.

Postpartum Thyroiditis

Postpartum thyroiditis may occur up to 12 months after delivery. Usually it presents as transient hyperthyroidism (median time of onset, 13 weeks post-delivery), followed by transient hypothyroidism (median time of onset, 19 weeks post-delivery). In the majority of patients, restoration of normal thyroid function occurs. Pregnancy triggers hormonal changes and immune system alterations, such as a shift from Th1 to Th2 cytokine production followed by a "rebound" shift back to Th1 after delivery, and fluctuations in transforming growth factor-beta1 TGF-β1) serum levels (29,30).

Anti-TPO and anti-Tg antibodies are found in almost all patients with postpartum thyroiditis. Notably, up to 50% of women who had anti-TPO antibodies at the end of the first trimester of gestation (i.e., before thyroid antibody titers start to decline during pregnancy) developed postpartum thyroiditis. Furthermore, there is evidence that the anti-TPO antibody titer at 16 weeks of gestation is related to the severity of postpartum thyroiditis (31), while activation of the complement is also involved in the pathogenesis (23).

Euthyroid Sick Syndrome

Euthyroid sick syndrome (ESS) is a condition characterized by alterations in thyroid hormone levels despite normal thyroid gland function. The characteristic laboratory abnormalities of the ESS include low T3 and/or fT3, elevated reverse T3 (rT3), normal or low TSH, and normal or low serum T4 or fT4 concentrations. Clinical conditions that trigger the development of ESS include systemic inflammation, myocardial infarction, starvation, sepsis, surgery, trauma, chronic degenerative diseases, malignancy, and every other condition associated with severe illness.

During illness or periods of severe physiological stress, the immune system releases various proinflammatory cytokines, such as in IL-6, TNF-α, IL-1β, IFN-γ, and TGF-β2. These cytokines disrupt the hypothalamic-pituitary-thyroid axis and interfere with the normal synthesis, secretion, and metabolism of thyroid hormones (Figure 3), while the more severe the illness, the more extensive the hormonal alterations.

Figure 3. Secretion of proinflammatory cytokines during severe illness or stress inhibits the activity of hepatic deiodinase type 1- suppressing the peripheral conversion of T4 to T3. They also suppress intrathyroidal hormone synthesis, TRH release and TSH secretion from the pituitary. IL-6, interleukin-6; IL-1, interleukin-1; TNF-α, tumor necrosis factor alpha; INF-γ, interferon gamma; TGF-β, tumor growth factor beta; TG, thyroglobulin; NIS, sodium/iodide symporter; TSH, thyroid stimulating hormone; TRH, Thyrotropin-releasing hormone.

Proinflammatory cytokines suppress the peripheral conversion of T4 to T3, resulting in low T3 levels and increase rT3 levels, by inhibiting the activity of hepatic deiodinase type 1, which promotes conversion of T4 to T3 and of rT3 to diiodothyronine (32). They also suppress TRH release and inhibit TSH response to TRH stimulation, leading to a decrease in TSH levels. Thus, the cause of the decreased T3 concentration in ESS is decreased T3 production, whereas the cause of the increased rT3 concentration is the result of impaired degradation. Prolonged and severe illness is marked by a decrease in circulating total T4 along with low T3 and high rT3, with very low T4 levels displaying a poor prognosis and having been associated with an increased mortality rate (32). In addition, cytokines reduce iodine uptake by inhibiting sodium-iodine symporter protein expression (25-27), and decrease thyrocyte growth (33), iodide organification (34,35), and thyroglobulin synthesis (36,37).

The central role of cytokines in the pathophysiology of ESS has been further elucidated in studies involving cytokine administration to humans. TNF-α administration in healthy volunteers caused a decrease in serum T3 and an increase in serum rT3 concentration (38). Unlike IL-6, serum TNF-α levels did not correlate with any of the typical thyroid parameters, such as low T3, increased rT3, or decreased TSH levels seen in ESS (39,40), suggesting that the changes of thyroid hormone profile following TNF-α administration might be indirect (i.e., through TNF-α increase in circulating IL-6 levels). Furthermore, both IL-6 and TNF-α can upregulate type 2 deiodinase in the anterior pituitary, affecting TSH release and contributing to the development of the non-thyroidal illness syndrome (41,42).

Leptin, a hormone primarily known for its role in regulating appetite and energy balance, has also been implicated in the development of EES. During acute illness or chronic inflammation, leptin levels are usually elevated. A link between leptin and the proinflammatory cytokines TNF-α and IL-6 in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (43) and ankylosing spondylitis (44), respectively, has also been proposed previously.

The primary effect exerted by leptin on the hypothalamic-pituitary-thyroid axis is alteration of the setpoint for feedback sensitivity of hypophysiotropic TRH-producing neurons in the paraventricular nucleus of the hypothalamus to thyroid hormones (mainly T3) by lowering of the setpoint when leptin levels are suppressed during fasting (45). Two anatomically distinct and functionally antagonistic populations of neurons in the arcuate nucleus of the hypothalamus, α-melanocortin-stimulating hormone (α-MSH)-producing neurons that co-express cocaine- and amphetamine-regulated transcript and neuropeptide Y (NPY)-producing neurons that co-express agouti-related peptide (AGRP), are responsible for the effects of leptin on hypophysiotropic TRH. It has also been proposed that leptin directly affects hypophysiotropic TRH neurons (46). Leptin has been found to inhibit the conversion of T4 to T3 in peripheral tissues and increase the activity of the enzyme type 3 deiodinase, which converts T4 to rT3. These data suggest that leptin can disturb thyroid function in seriously ill patients via two different independent mechanisms (cytokine-dependent and directly).

Amiodarone-Induced Thyroid Disease

Amiodarone, a benzofuran derivative with a similar structure to that of thyroid hormones, is a highly effective antiarrhythmic agent widely used in the treatment of various types of tachyarrhythmias (supraventricular and ventricular arrhythmias). Amiodarone contains two iodine atoms per molecule, which is approximately 37.5% iodine by molecular weight (47).

Treatment with amiodarone may be related to an increase in lymphocyte subsets leading to an exacerbation of pre-existing autoimmunity (48,49). The relative proportion of patients developing either thyrotoxicosis or hypothyroidism depends on the iodine content of the local diet and pre-existing thyroid autoimmunity. In relatively iodine-replete areas, approximately 25% of patients with amiodarone-induced thyroid dysfunction become thyrotoxic, accounting for approximately 3% of amiodarone-treated individuals (50).

Amiodarone-induced hypothyroidism is attributed to an increased susceptibility to the inhibitory effect of iodide on thyroid hormone synthesis and/or to a failure to escape the Wolff-Chaikoff effect (49). Hashimoto thyroiditis is the most common risk factor for amiodarone-induced hypothyroidism and it is considered the most likely reason for the female preponderance of this clinical entity (51). Female patients with positive anti-TPO and anti-Tg autoantibodies have a relative risk of 13.5% for developing amiodarone-induced hypothyroidism compared to men without thyroid autoantibodies (20).

The pathogenesis of amiodarone-induced thyrotoxicosis is complex, although two distinct forms, type 1 and type 2, are recognized. Type 1 develops in patients with latent thyroid disease, predominantly nodular goiter, in whom the amiodarone iodine load triggers increased synthesis of thyroid hormones. Type 2 is the result of a destructive thyroiditis in a previously normal gland, with leakage of preformed thyroid hormones despite a reduction in hormone synthesis (47,50,52,53).

Differentiating between the two types of amiodarone-induced thyrotoxicosis is an essential step in their management, as treatment of each type is different (50). Type 1 usually responds to thionamide therapy, which blocks hormone synthesis, and perchlorate, which blocks active transport of iodine into the thyroid, whereas type 2 responds to high-dose glucocorticoids (50,53-55). Nevertheless, several studies now suggest that these two types should be treated concomitantly; thus, currently, patients with amiodarone-induced thyrotoxicosis receive both antithyroid drugs and prednisolone. In cases resistant to medical treatment and/or in patients with severe cardiac diseases who cannot interrupt amiodarone or require quick amiodarone reintroduction, total thyroidectomy may be offered after rapid correction of thyrotoxicosis following combination treatment with thionamides, KClO4, glucocorticoids, and a short course of iopanoic acid (56).

Thyroid Cancer

Thyroid cancer is the most common endocrine cancer, the incidence of which has steadily increased over the past few decades (57).

The association of chronic inflammation induced by Hashimoto thyroiditis and thyroid cancer has been long recognized (58). However, the immune response triggered against thyroid cancer and AITDs differs significantly. In thyroid cancer, the immune response is more tolerant and allows tumor growth, whereas in AITDs, the response is more aggressive, triggering cell destruction and reduction of the function of the gland (59). Hashimoto thyroiditis is considered both a risk factor for the development of thyroid cancer (60) and a favorable prognostic factor due to chronic lymphocytic infiltration, which can downregulate tumor aggressiveness (60,61). In Graves’ disease, the presence of a strong humoral immune response appears to be protective against thyroid cancer. Patients with increased anti-TPO and anti-Tg levels show lower distant metastasis rates than patients without thyroid autoantibodies (62,63), suggesting their potentially protective role (59).

Immune infiltrates in the tumor microenvironment differ between the different thyroid neoplasm subtypes (Figure 4). In general, differentiated thyroid cancer (DTC) has a higher number of tumor-associated lymphocytes and regulatory T cells (Tregs), while anaplastic thyroid cancer (ATC) and medullary thyroid cancer (MTC) display a high density of tumor-associated macrophages (64). The number of tumor-associated macrophages has been associated with dedifferentiation, lymph node metastases, and reduced survival (65). It is important to note, however, that most of the studies analyzing the immune milieu of DTC have used papillary thyroid cancer (PTC) tumor samples (66-69). Myeloid-derived suppressor cells are associated with aggressive characteristics of differentiated thyroid cancer and are related to poor prognosis (65).

Figure 4. Immune infiltrates differ in different types of thyroid cancer [Modified by Garcia-Alvarez et al, (70)]. TAM, tumor-associated macrophages; MDSC, myeloid-derived suppressor cell.

The dendritic cells, which play a critical role in antigen presentation, are increased in PTC, while neutrophils are found in more aggressive thyroid cancers (such as poorly differentiated or anaplastic). In addition, natural killer cells that play an important role in immunosurveillance are also increased in PTC and are negatively correlated with tumor stage, while lymphocytic infiltration is associated with better overall survival and low recurrence rate (71,72).

Cytokines, which may be produced by thyroid follicular cells and by immune cells infiltrating thyroid tumors, are also related to tumor development. IL-1 and IL-6 stimulate thyroid cell proliferation and tumor growth, while TGF-β, which is a suppressive cytokine, is overexpressed in aggressive cancers (73). In addition, multiple chemokines may be secreted by thyroid cancer or immune cells and affect chemiotaxis, angiogenesis, and lymphangiogenesis (73).

Immunomodulatory proteins, such as programmed death-ligand 1 (PDL1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin and ITIM domain (TIGIT), which are considered major immune coinhibitory receptors and promising immunotherapeutic targets in cancer treatment, are also expressed in thyroid cancer, being associated with more aggressive tumor characteristics and a poor prognosis (64). Programmed death-ligand 1 staining by immunohistochemistry has shown higher expression in ATC than in other subtypes (70). Six cohort studies have been published to-date reporting positive PD-L1 expression, varying between 22% and 65%, this being higher compared to that detected in DTC and poorly differentiated thyroid cancer (74-79). TIM-3 expression was observed in 48% of patients with MTC, and in the majority of cases (84.4%) its expression was restricted to tumor cells (80). Other coinhibitory receptors, such as LAG-3 and T cell TIGIT, were observed in a lesser percentage of cases (approximately 3%) (80). Nevertheless, results from clinical trials with immunotherapy as monotherapy or combinations have shown limited efficacy (70). In one phase Ib KEYNOTE-028 trial assessing the efficacy of pembrolizumab in patients with PD-L1+ (membranous staining on ≥1%) locally advanced or metastatic follicular or papillary thyroid cancer, pembrolizumab achieved an objective response rate of 9% and a median progression-free survival of 7 months (81). Several clinical trials further investigating the efficacy of combination therapy of immune checkpoint inhibitors are currently ongoing.

IMMUNE SYSTEM AND DIABETES MELLITUS

The immune system plays a crucial role in the pathogenesis of both type 1 and type 2 diabetes.

Diabetes type 1, which is immune-mediated in more than 95% of cases, is an organ-specific autoimmune disease characterized by lymphocytic infiltration and inflammation that leads to pancreatic β-cell destruction and absolute insulin deficiency (82). The immune system’s attack on pancreatic β-cells is usually triggered by a number of factors, including genetic predisposition and environmental triggers such as viral infections (e.g., Coxsackie B4, mumps, and rubella) or dietary compounds (e.g., cow’s milk) (82). The process involves the activation of immune cells, particularly T cells, which recognize self-autoantigens in pancreatic β-cells and initiate an immune response.

On the other hand, in type 2 diabetes, the immune system also plays a different role from that in DM1. Chronic low-grade inflammation, often associated with obesity, leads to immune cell activation and the release of proinflammatory cytokines. These cytokines interfere with the normal functioning of insulin and promote insulin resistance. Macrophages infiltrate adipose tissue and release inflammatory molecules, further exacerbating insulin resistance with increasing adiposity. The immune system, thus, contributes to the development and progression of insulin resistance and eventually promotes the onset of DM2.

Understanding the intricate relations between the immune system and diabetes pathogenesis is essential for the development of effective treatments and interventions for the management of both types of diabetes.

Diabetes Type 1

The susceptibility to develop DM1 is associated with multiple alleles of the major histocompatibility complex MHC I and II locus. More than 90% of patients with DM1 express either HLA DR3, DQ2 or DR4, or DQ8 (83), whereas HLA haplotype DR2, DQ6 is protective against DM1 development.

The primary pathological presentation of DM1 is inflammation of the pancreatic islets, also known as insulitis, caused by infiltration of immune cells, including CD4 and CD8 T cells along with B cells (84-86). Although the initial events triggering autoreactive responses remain unclear, presentation of pancreatic islet autoantigens by the associated MHC class II molecules contribute to priming and expansion of pathogenic T cells.

CD4 T helper cells are required for the development of the autoimmune process in the pancreatic islets, while CD8 cytotoxic T cells are the cells responsible for β-cell destruction (Figure 5). T cell receptors recognize peptides bound to MHC molecules on the surface of antigen-presenting cells (B-cells, dendritic cells, and macrophages). Each T cell then generates a unique receptor for the recognition of an autoantigen presented in the MHC molecule. The interaction between T cell receptor/autoantigen/MHC leads to activation of the T cells.

Figure 5. T cell mediated destruction of β- cells in diabetes type 1. CD4+ effector T cells recognize an insulin or islet peptide presented by diabetes-risk conferring HLA-DQ8 or DR4 on APCs, migrate to the pancreas and promote pancreatic β-cell destruction. Inside the pancreatic islets CD8+ effector T cells recognize islet antigens presented by HLA-A2 leading to the destruction of the β-cells. [Modified by Mitchel et al (87)]. TCR, T cell receptor; APC, antigen presenting cell; HLA-A2, human leukocyte antigen-A2.

Three different mechanisms have been proposed to explain T cell activation in DM1. One mechanism is thought to involve molecular mimicry-activated T cell proliferation. The hypothesis for this mechanism is based on the assumption that epitopes of proteins expressed by infectious agents can be shared by unrelated molecules encoded by host genes (88). A second mechanism that may trigger molecular mimicry-activated T cell proliferation is “bystander” T cell proliferation. This mechanism involves the stimulation of non-antigen-specific T cells by various cytokines during infection, simply because they are in the area. The cytokines thought to be involved in this nonspecific stimulation are IFN-α and IFN-β (89). The 3rd mechanism might involve a superantigen-mediated T cell proliferation mechanism: this theory proposes that autoreactive T cells can be inappropriately primed to react against self-structures through an encounter with a superantigen (90).

Multiple autoantigens in the pancreatic islets have been identified, including non-specific islet cell autoantigens (ICA), insulin, glutamic acid decarboxylase 65 (GAD65), insulinoma antigen-2 (IA-2), heat shock protein (HSP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), imogen-38, zinc transporter-8 (ZnT8), and the most recently identified pancreatic duodenal homeobox factor 1 (PDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP) (91). Autoantibodies against these autoantigens may be detected long before the onset of hyperglycemia and usually decline during the course of the disease (92).

Most patients diagnosed with DM1 have circulating islet cell autoantibodies directed against pancreatic islet autoantigens. However, although the detection of autoantibodies may be useful for DM1 diagnosis and prediction, it is the cellular immune system that eventually infiltrates the pancreatic islets and causes β-cell destruction. In turn, further β-cell destruction leads to more self-antigen presentation and ensuing amplification of the immune response (82,93,94).

Among several proinflammatory cytokines, IL-21, produced by CD8 T cells, is required for the development of DM1, while TNF-α may also be involved (95,96). IL-6 plays an important role in the pathogenesis of vitiligo-associated DM1 (97). In contrast, in vivo experiments with non-obese diabetic mice have shown that IL-4, produced by Th2 cells, may be protective against developing diabetes (95,96,98,99).

Understanding both the pathophysiology and the regulatory mechanisms involved in DM1 is a critical step towards the development of antigen-specific, β-cell-directed, immunomodulatory or cellular treatment modalities (100).

Investigations into the efficacy and safety of various immunotherapeutic strategies against the development of DM1 have been carried out in recent clinical trials and are still ongoing in current trials (101). Among them, T cell-directed therapies that aim at a favorable balance between effector T cell depletion and regulatory T cell preservation have shown the most promising results. Teplizumab, an anti-CD3-directed monoclonal antibody, was the first immunomodulatory agent to demonstrate a significant delay in disease progression in high-risk individuals before clinical onset (102) and has recently (November 17, 2022) been approved by the FDA as the first disease-modifying therapy for DM1 in adults and in children aged 8 years and older.

The Enigma of Pancreatic α-Cell Resistance in Diabetes Mellitus Type 1

Although both insulin-producing β-cells and glucagon-producing α-cells of the pancreatic islets share a similar embryonic origin and are directly exposed to the deleterious immune signals in DM1, it appears that the immune system selectively destroys β-cells, while α-cells survive even in long-term DM1 (103). α-Cells are located in close proximity to β-cells in human pancreatic islets (104), creating a closed communication loop that regulates their function and secretory capacity (105). Dysfunction of α-cells plays a significant role in the pathogenesis of both types of diabetes (106). It has long been proposed that the reduced functional β-cell mass in DM1 and the consequent hypoglycemia were the key mechanisms indirectly inducing dysfunction of α-cells in diabetes (107). Advanced molecular technology of the last decade has, however, challenged this notion, showing that dysfunction of α-cells in diabetes is not secondary to β-cell pathology but is instead directly immune-induced (108-110). Despite this dysfunction, α-cells exhibit a remarkable autoimmune resistance that enables them to survive over β-cells in long-standing diabetes (103,111). Recent studies using single-cell RNA sequencing (109,112) have demonstrated important differences between these two cell types in terms of expression of: i) anti-apoptotic genes, ii) endoplasmic reticulum (ER) stress-related genes, III) innate immune response genes, and iv) antiviral response genes, all of which render α-cells less immunogenic and more resistant to viral infections and ER stress (113). In addition, CD8+ T cells invading the islets in DM1 are reactive to preproinsulin but not to glucagon. Furthermore, although β-cells are essential to life (neither humans nor animal models can survive without them), mice with 98% α-cell ablation retain near-normal glucose homeostasis (114): this points to the knowledge gap we have, from an evolutionary point of view, regarding the question of why β-cells are more fragile than α-cells. Certainly, greater understanding of the underlying mechanisms responsible for the autoimmune resistance of α-cells is critical since it is likely to reveal intracellular pathways amenable to therapeutic interventions that would increase the resistance of the β-cells themselves to the immune attack of the host’s immune system.

Diabetes Type 2

DM2 accounts for 90% of cases of diabetes worldwide (115). The increasing prevalence of DM2 around the world has been largely attributed to an unhealthy lifestyle and resultant development of obesity and overweight (116). Obesity is strongly related to DM2 mainly through inducing insulin resistance in the insulin sensitive tissues in the periphery.

The concept that a smoldering inflammatory process plays an important part in the pathogenesis of DM2 (117) has attracted much attention and is supported by evidence of inflammation in islets, adipose tissue, liver, and muscle that can provoke insulin resistance and β-cell dysfunction (118-120). Adipose tissue is characterized by infiltration by macrophages and other immune cells that produce cytokines and chemokines and contribute to the development of local and systemic chronic low-grade inflammation, this inflammatory milieu being the link between obesity, insulin resistance, and diabetes mellitus (121,122).

Earlier in vivo studies demonstrated that levels of TNF-α (123-125), IL-6, C-reactive protein, plasminogen activator inhibitor, and other inflammation mediators were elevated in adipose tissue and plasma of obese mice (126,127). It was also observed that these inflammatory mediators, together with saturated free fatty acids and reactive oxygen species (ROS), inhibited serine phosphorylation of the insulin receptor substrates (IRS-1 and 2) (128-130) in insulin sensitive tissues, such as adipose tissue and the liver (131), promoting insulin resistance (Figure 6). TNF-α is associated with increased release of free fatty acids by adipose tissue and leads to impaired insulin secretion and signaling (123,132).

Figure 6. Insulin resistance and inflammation in diabetes type 2. Insulin binds to insulin receptor (IR) in insulin sensitive tissues, and autophosphorylates tyrosine molecules of IRS-1 and -2 substrates. In the presence of obesity, and hyperlipidemia, the influx of free fatty acids, inflammatory cytokines and glucose activates IKKβ and JNK, which are the mediators for stress and inflammatory. In turn IKKβ and JNK inhibit tyrosine phosphorylation of IRS1 and 2 and promote transcriptional activation of genes related to inflammatory and stress responses resulting in insulin resistance. [Modified by Berbudi et al (133)]. IRS 1 -2, insulin receptor substrates; ER, Endoplasmic reticulum; IKKβ, inhibitory kappa B kinase β; JNK1 and c-Jun N-terminal kinase I.

In line with these early in vivo studies, studies in humans have shown that elevated levels of i) nonspecific indicators of inflammation, such as white cell count, fibrinogen, and CRP levels (134-136), ii) markers of reduced fibrinolysis, such as plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA), iii) von Willebrand factor (vWf), which is a marker of endothelial injury, and iv) early markers of inflammation, such as monocyte chemotactic protein-1 (MCP-1), IL-8, and interferon-γ-inducible protein-10 (137), were predictive of DM2 development.

Furthermore, CRP and/or IL-6 were associated with the incidence of DM2 independently of adiposity or insulin resistance (136,138,139). Visceral adipose tissue appears to be a major source of circulating IL-6 in humans, and obese people with insulin resistance display high levels of plasma IL-6 concentration, also predictive of DM2 development (113). TNF-α is also increased in obese individuals with insulin resistance (124,140) and it plays a major role in the pathogenesis of obesity-linked DM2 (141).

At the cellular level, chronic exposure of adipocytes to low doses of TNF-α led to a dramatic decrease in insulin-stimulated auto-phosphorylation of the IRS 1-2 (142). Treatment of cultured murine adipocytes with TNF-α induced serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. TNF-α has also been shown to downregulate glucose transporter GLUT4 mRNA levels in adipocyte and myocyte cultures as well (125,143,144).

Oxidative stress, as a result of increased cytokine levels in DM2, is also thought to play an important role in activating inflammatory genes (145,146) (Figure 6). It is possible that oxidative stress markers do not adequately reflect the impact of increased ROS on β-cells or insulin signaling, while inflammatory, procoagulant or endothelial dysfunction markers are more specific to the pathophysiology of hyperglycemia (145,146). Hasnain et al. showed that islet-endogenous and exogenous IL-22 suppressed oxidative and ER stress caused by cytokines or glucolipotoxicity in mouse and human β-cells by regulating oxidative stress pathways. In obese mice, antibody neutralization of IL-23 or IL-24 partially reduced β-cell ER stress and improved glucose tolerance, whereas IL-22 administration modulated oxidative stress regulatory genes in islets, suppressed ER stress and inflammation, promoted secretion of high-quality efficacious insulin, and fully restored glucose homeostasis, followed by reinstitution of insulin sensitivity (147).

The chemokine system is also associated with obesity and insulin resistance. MCP-1, which acts on monocytes, macrophages, T cells and NK cells, is increased in obese compared to lean subjects and is related to non-alcoholic fatty liver disease and other lipid overload states (148-153). A short-term program of 4-month lifestyle modification significantly decreases MCP-1 levels, with favorable effects on the glycemic and lipid profile (151).

Collectively, these findings supported the investigation of new therapeutic approaches that target inflammation to ameliorate diabetes and its complications. Regarding the latter, it is important to note that multiple sources of evidence support a pathogenic connection between rheumatoid arthritis (RA) and the mechanisms of DM2, via formation of a vicious circle that is perpetuated by impaired glucose metabolism and inflammation. In this context, ongoing clinical studies have shown that the inhibition of interleukin IL-1 and IL-6 may allow the treatment of RA and concomitant T2D at the same time (154).

Treatment with anakinra, a recombinant form of human IL-1 receptor antagonist that works as a competitive inhibitor of IL-1β, achieved significant improvements of glycemia and secretory function of β-cells (155,156), which was maintained after anakinra withdrawal during a 39-week follow-up (157). Canakinumab, a monoclonal antibody against IL-1β, significantly reduced inflammatory proteins, such as CRP, IL-6, and fibrinogen, in patients with DM2 and high cardiovascular risk (158,159), while regarding glycemic control, it reduced values of HbA1c during the first 6–9 months of treatment, without, however, consistent long-term benefits (159). Antagonists of IL-6 receptor tocilizumab and sarilumab have also been investigated in patients with RA with or without concomitant DM2. Tocilizumab showed a positive effect on insulin resistance in some studies (160-162), while other studies failed to report any beneficial effect on glycemic control (163,164). The efficacy of sarilumab was assessed in a post hoc analysis (165) of three randomized clinical trials in patients with RA with or without DM2 (166-168). Sarilumab, as monotherapy or in combination, significantly reduced HbA1c compared to adalimumab monotherapy or placebo plus methotrexate/convectional DMARDs in patients with RA and DM2 (165).

Several studies have analyzed the effects of TNF inhibitors (i.e., adalimumab, etanercept, and infliximab) on glucose metabolism, demonstrating a potential favorable effect on insulin resistance and insulin sensitivity (169,170). In a meta-analysis combining data from 22 randomized controlled trials and three cohort studies (171), new-onset DM2 was delayed in RA patients treated with TNF inhibitors.

The Effect of Diabetes on the Immune System

Like a mirror image, chronic hyperglycemia in diabetic patients impairs the host’s immune response, which in turn fails to control the spread of invading pathogens, rendering diabetic patients more susceptible to infections (133,172). Both innate immune response defects and defective adaptive immune response are implicated in this incapacity of the immune system to defend against invading pathogens in patients with diabetes. Several mechanisms have been proposed by experimental and human studies including: i) leukocyte recruitment inhibition (173,174), ii) defective pathogen recognition (175), iii) dysfunction of neutrophils (176-178), macrophages resulting in impairment of phagocytosis (179), iv) functional defects in natural killer cells (180), and v) dysfunction of complement activation (181).

OSTEOPOROSIS AND THE IMMUNE SYSTEM

Osteoporosis is a clinical condition characterized by low bone mass and impaired bone microarchitecture associated with an increased risk of fragility fractures. Growing evidence of the last few decades has demonstrated the effect of the immune system on bone metabolism, leading to the emergence of the new field of osteoimmunology (182-185) and immunology of osteoporosis (named as immunoporosis) (184-188).

States of immune dysfunction such as immunodeficiency, inflammatory diseases, or immune response to infections are associated with increased osteoclastic bone resorption and, therefore, increased bone loss and increased fracture risk.

Bone Remodeling and Bone Cells

Bone remodeling is a dynamic and continuous process that is responsible for the maintenance of skeletal health throughout life. It involves three consecutive phases: i) osteoclast-mediated bone resorption; ii) the reversal phase, during which mesenchymal derived osteoblasts are recruited to the bone site of bone resorption; and iii) osteoblast-mediated bone formation. The two processes of bone resorption and bone formation are tightly coupled and under control of the matrix-embedded osteocytes, capable of sensing and integrating mechanical and chemical signals from their environment to regulate bone formation and resorption at the bone surfaces.

Osteoclasts originate from the same myeloid precursor that derives macrophage and dendritic cells and are specialized in bone degradation (186). Osteoblasts are the main bone-forming cells and are derived from mesenchymal stem cells. Osteoclast formation and differentiation is regulated by macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) produced by osteoblasts and osteocytes (189,190). Osteocytes are a significant source of RANKL and its decoy receptor, osteoprotegerin (OPG), which binds to RANKL, preventing its interaction with RANK, while they also secrete the Wnt signaling inhibitor sclerostin, which regulates bone formation (189). RANKL is additionally expressed by fibroblasts and immune cells, including antigen-stimulated T cells and dendritic cells (191-193), while OPG is also produced by B lymphocytes and dendritic cells (194).

Inflammatory Diseases

Activated T cells increase the production of TNF-α and RANKL and stimulate osteoclastogenesis during inflammation (182-185,192-195). Multiple cytokines may promote osteoclastogenesis mainly by regulating the RANK/RANKL/OPG axis. TNF-α, IL-1, IL-6, IL-7, IL-11, Il-17, and IL-23 promote osteoclast differentiation, while IFN-α, IFN-γ, IL-3, IL-4, IL-10, IL-27, and IL-33 are considered anti-osteoclastogenic cytokines that protect bone integrity (195). Th17 cells are considered an osteoclastogenic subset of T cells as they enhance osteoclastogenesis by secreting IL-1, IL-6, Il-17, RANKL, TNF-α, and IFN-γ. Activation of Th2 leads to enhanced production of PTH and promotes the anabolic activity of osteoblasts in several inflammatory states. Furthermore, Th2 lymphocytes are associated with a low RANKL/OPG ratio and inhibition of bone loss (196). In addition, β-cells produce RANKL and OPG and may influence bone formation and absorption, while it has been observed that in HIV infected patients, there is an altered β-cell RANKL/OPG ratio that is inversely correlated with BMD (197).

Interleukin 6, produced by both stromal and osteoblastic cells (198) in response to stimulation by systemic hormones such as PTH, PTH-related peptide (PTH-rP), thyroid hormones, and 1,25-dihydroxyvitamin D3 and other cytokines (i.e., TGF-β, IL-1, and TNF-α), plays a major role in osteoclast development and function. Increased IL-6 levels contribute significantly to the abnormal bone resorption observed in patients with multiple myeloma (199), Paget’s disease of bone (200), rheumatoid arthritis (201), and Langerhans cell histiocytosis (202). Effects of increased osteoclast-induced bone resorption are not solely attributable to IL-6, but to all IL-6 family cytokines (203).

TNF-α has also been shown to induce bone resorption and plays an important part in inflammatory bone diseases (192). TNF-α promotes RANKL expression in osteoclast precursors and the formation of multinucleated osteoclasts in the presence of M-CSF. Furthermore, TNF-α increases RANKL and M-CSF expression in osteoblasts, stromal cells, and T lymphocytes, while RANKL can also enhance TNF-α mediated osteoclastogenesis (195). IL-1β increases RANKL expression and stimulates osteoclast formation and bone resorption while also promoting TNF-α induced osteoclastogenesis (204,205).

Postmenopausal Osteoporosis

Estrogen deficiency is a state of increased bone remodeling associated with an increased rate of bone resorption relative to bone formation, resulting in net bone loss. It has been shown that estrogen deficiency-induced bone loss has a complex mechanism mainly involving the immune system rather than a direct effect of estrogen on bone cells (206). Estrogen loss during menopause is associated with an expansion of T and B lymphocytes (207,208) leading to increased production of RANKL (209). In addition, an increased level of proinflammatory cytokines, including TNF-a and IL-1b, is observed in postmenopausal women (210,211). In addition, B lymphocytes may partially contribute to trabecular bone loss in postmenopausal osteoporosis (212). In the absence of estrogens, dendritic cells live longer, increasing their expression of IL-7 and IL-15. In turn, IL-7 and IL-15 induce IL-17 and TNF-a production in a subset of memory T cells, independent of antigen activation (213). These proinflammatory cytokines contribute to inflammation-induced bone loss in postmenopausal osteoporosis by activating low-grade inflammation. In contrast, Treg cells have a bone-protective role in postmenopausal osteoporosis (210). However, it has been shown that Th17/Treg balance is disturbed in estrogen deficiency. Treg cells lose their immunosuppressive function and convert to Th17 cells, which explains the imbalance of Th17/Treg in postmenopausal osteoporosis (214).

Senile Osteoporosis

Senile osteoporosis, on the other hand, is a low-bone turnover disease with decreased bone resorption and significantly reduced bone formation (215), which commonly occurs in older people, above 65, and affects both males and females. In recent years, it was demonstrated that aging is usually accompanied by systemic low-grade chronic inflammation and enhanced inflammatory mediators, such as IL-6 and TNF-a (216). A recent study found that senescent immune cells, such as macrophages and neutrophils, accumulate in bone marrow during aging in rats and mice (217). The senescent macrophages and neutrophils repress osteogenesis by promoting bone marrow mesenchymal stromal cell adipogenesis. In addition to directly inhibiting osteogenesis, the senescent immune cells contribute to chronic inflammation, thus leading to inflammatory bone resorption (217). Aging can tilt the balance of Th1/Th2 toward Th2 cells, resulting in an increased inflammatory response (218) and low-level chronic inflammation, ultimately leading to continuous bone loss.

Thyrotoxicosis-Induced Osteoporosis

IL-6 and IL-8 play a major role in thyrotoxicosis-induced osteoporosis and are increased in patients with thyrotoxicosis due to Graves’ disease or toxic multinodular goiter (219). In addition, patients with thyroid carcinoma on TSH suppressive therapy have significantly increased circulating levels of IL-6 and IL-8 compared to controls (219), which are tightly associated with serum T3 and fT4 concentrations. Both IL-6 and IL-8 have also been shown to be released by human bone marrow stromal cell cultures containing osteoblast progenitor cells in response to T3 (196). TNF-α elevations due to low TSH signaling in human hyperthyroidism also contribute to the bone loss that has traditionally been attributed solely to high thyroid hormone levels (220). Hyperthyroid mice lacking TSHR had greater bone resorption than hyperthyroid wild-type mice, demonstrating that the absence of TSH signaling contributes to low bone mass (221) in the hyperthyroid state.

Primary Hyperparathyroidism-Induced Osteoporosis

Bone resorption in primary hyperparathyroidism (PHP) also appears to be related to immune system effects. Circulating levels of IL-6 and TNF-α, which are significantly increased in patients with PHP, are strongly correlated with biochemical markers of resorption, returning to normal after successful parathyroidectomy (222). The hypothesis that IL-6 mediates the catabolic effects of parathyroid hormone (PTH) on the skeleton has been further strengthened by the finding that neutralizing IL-6 in vivo attenuates PTH-induced bone resorption in mice, while the resorptive response to PTH was also reduced in IL-6 knockout mice (223). Furthermore, it has been observed that transplantation of parathyroid from humans with hyperparathyroidism to mice lacking T cells was not associated with bone loss, suggesting a possible role of T lymphocytes in PTH-related osteoporosis (224). A direct action of PTH on T lymphocytes may also contribute, as deletion of the PTH receptor from T cells failed to induce bone loss (225). It has been proposed that PTH action on T cells results in secretion of TNF-α and, in combination with RANKL increase and OPG suppression, guides their differentiation to Th17 subsets, with subsequent IL-17 secretion and further RANKL amplification (226).

Drug-Induced Osteoporosis

The immune cells are also involved in drug-induced osteoporosis, such as glucocorticoid and chemotherapy-induced osteoporosis. A recent study demonstrated that glucocorticoid-induced osteoporosis could not be induced in T cell-deficient mice, while re-establishment was found to be possible by transferring splenic T cells from wild-type mice (227).

Cyclophosphamide, is a chemotherapy drug that causes immunosuppression and is associated with increased risk of osteoporosis (228-230). By improving the functional status of immune cells in an immunosuppressive mouse model induced by cyclophosphamide, bone loss was dramatically reduced (231), pointing to the possible contribution of immune cells in cyclophosphamide-induced osteoporosis.

EFFECTS OF THE IMMUNE SYSTEM ON THE STRESS SYSTEM

The Hypothalamic-Pituitary-Adrenal (HPA) Axis

The relations between the immune and the stress systems are complex and bidirectional, denoting that while stress can affect immune function, immune responses can also influence stress levels through various ways and mechanisms.

INFLAMMATION

During acute inflammation, the immune system is activated in response to infection or injury and releases proinflammatory cytokines and other inflammation-related factors into the central nervous system (CNS), plasma, and endocrine glands. Inflammatory cytokines, such as TNF-α, IL-1, and IL-6, produced by a variety of cells, including monocytes, macrophages, astrocytes, endothelial cells, and fibroblasts, activate the HPA axis leading to an increase of corticotropin-releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and, finally, glucocorticoids (38, 184). In turn, increased circulating levels of glucocorticoids exert suppressive effects on the inflammatory reaction, controlling the immune response and helping the organism to reach its prior healthy homeostasis (232). Similarly, in acute stress, the amplitude and synchronization of CRH secretory pulses is increased, and this is reflected in the levels of ACTH and cortisol in the systemic circulation (233).

The proinflammatory cytokine IL-1, especially its β form, is probably the most important molecule capable of modulating cerebral functions during systemic and localized inflammation. Systemic IL-1β injection activates the neurons involved in the control of autonomic functions, and neutralizing antibodies or IL-1 receptor antagonists are capable of preventing numerous responses during inflammatory stimuli (234). Similarly to IL-1β, intravenous IL-6 stimulates the hypothalamic-pituitary unit, leading to the secretion of cortisol by the adrenal glands and subsequent termination of the inflammatory cascade (235). All three inflammatory cytokines (IL-1, IL-6, and TNF-α) have the capacity to activate the HPA-axis, but it appears that IL-6 is the most critical component of this cascade. Studies in rats have demonstrated that immunoneutralization of IL-6 abolishes the effects of the other two cytokines on the HPA-axis (236). TNF-α and IL-1, on the other hand, stimulate the production of IL-6, which in turn stimulates the HPA-axis. The final end-product of HPA activation, glucocorticoids, inhibit IL-6 secretion at the transcriptional level through interaction of the ligand-activated glucocorticoid receptor with nuclear factor-kappa B, creating a negative feedback loop. In this way, IL-6 stimulates glucocorticoid release to control inflammation, and glucocorticoids subsequently inhibit IL-6 release through a negative feedback loop preventing uncontrolled and potentially harmful sequalae of inflammatory mechanisms, including tissue damage (237,238).

In an older study involving patients with Cushing disease studied before and after transsphenoidal adenectomy, plasma IL-6 concentration was increased when patients were hypocortisolemic, experiencing symptoms of glucocorticoid deficiency, as part of the “steroid withdrawal syndrome” (i.e., pyrexia, headache, anorexia, nausea, fatigue, malaise, arthralgias, myalgias, and somnolence of variable degree). Notably, IL-6 levels did not increase in patients who did not become hypocortisolemic after surgery, while following glucocorticoid replacement, a dramatic decrease of IL-6 levels concomitantly with relief of the observed symptoms was reported (239).

While acute stimulation with IL-6 activates the HPA axis mainly through the hypothalamic CRH neurons, chronic exposure to IL-6 may also directly stimulate the pituitary corticotropic cells and the adrenal cells via CRH receptor-independent mechanisms (240).

In vivo experiments have shown a stimulatory effect of IL-6 on cortisol production by the adrenal cortex in the absence of CRH and subsequent activation of the HPA axis (240) in cytomegalovirus-infected mice (241), as well as in murine colitis (242). In addition, experiments in mice deficient in CRH (CRH KO) and deficient in both CRH and IL-6 (CRH KO)/IL-6 KO) have demonstrated that IL-6 during prolonged immunological challenge may surpass CRH in augmentation of adrenal function (240). Protein expression of IL-6 and IL-6 receptor has been detected in primary cultures of human adrenocortical cells depleted of macrophages (CD68-positive cells), predominantly in the zona reticularis but also in the zona fasciculata and in single cells within the zona glomerulosa and the medulla (243). Moreover, IL-6 was able to induce adrenal steroidogenesis in vitro in a time- and dose-dependent manner in the absence of macrophages, suggesting that IL-6 may be a long-term stimulator of steroidogenesis but with no acute effects (243).

In humans, IL-6 may stimulate cortisol release directly at the level of the adrenal gland in long-term stress situations (244), and the same may also apply in chronic inflammatory diseases, although direct evidence is still lacking (245). In a recent clinical study, increased daily, and especially evening, saliva cortisol secretion, in the context of the acute viral infection COVID-19, appeared to be mostly driven by hypersecretion of IL-6, independently of ACTH (246). However, the hypothesis that IL-6 may partially replace ACTH when there is an acute requirement for increased cortisol secretion has yet to be tested.

PROLONGED OR CHRONIC STRESS AND INFLAMMATION

Acute versus chronic stress and inflammation are distinct conditions that exert extremely different effects on the immune system, each altering its function in a distinct manner. Chronic stress is associated with a blunted circadian cortisol rhythm, a suppressed inflammatory response, and a shift from Th1 to Th 2 and a Th reg to Th 17 immunity. It has clearly been shown that in chronic stress, endogenous glucocorticoids fail to terminate the stress response and, in fact, cause body composition changes reminiscent of those in hypercortisolism, such as visceral adiposity, sarcopenia, and osteoporosis (247-249).

On the other hand, following a period of intense stress, there may be glucocorticoid-induced suppression of the HPA axis (238). In this case, long-term “inadequate” cortisol secretion may unleash its inhibitory effect on immune system activation, resulting in immune dysregulation and expression of sickness-syndrome manifestations. In this case, the post-stress sustained HPA axis suppression and hypocortisolism is reminiscent of the clinical picture of the intrinsic hypocortisolemia of primary adrenal insufficiency (Addison’s disease), associated with immune perturbations due to failure of cortisol to appropriately suppress the increased secretion of proinflammatory cytokines (239,250).

The subsequent protracted lack of axis recovery due to a post-illness state of hypocortisolism in probably predisposed individuals may explain the underlying pathophysiologic mechanisms responsible for some post-viral infection sickness syndromes (232,251), such as long COVID syndrome (252).

AUTOIMMUNE DISORDERS

In some cases, chronic stress can contribute to the development or exacerbation of autoimmune disorders. Without appropriate cortisol regulation in cases of chronic stress and chronic inflammation, the organism fails to downregulate inflammatory processes, contributing to a vicious cycle where stress, inflammation, and compromised HPA axis function may result in the development of chronic immune-mediated inflammatory diseases, such as rheumatoid arthritis. Stress can potentially trigger or worsen these conditions by dysregulating the immune response.

Cortisol diurnal secretion displays a circadian rhythm in healthy individuals, with the highest levels in the morning and a gradual decline throughout the day, reaching the lowest levels around midnight (253). During acute stress (254-256) or infection (237,257), the rhythm is disturbed and the afternoon and/or midnight cortisol levels do not drop. A disturbed circadian cortisol rhythm with abnormally high afternoon and night cortisol levels was found in patients with even mild or moderate COVID-19 compared to healthy controls (246). However, although not systematically studied, the adrenal response to chronic versus acute exposure to proinflammatory cytokines appears to follow a different pattern.

In patients with rheumatoid arthritis, symptoms follow circadian rhythms with impaired function due to pain and joint stiffness being most severe in the early morning (258,259) because of increased proinflammatory cytokines, such as TNF-a and IL-6, that occur during late night hours (260,261). This explains the inverse relation between diurnal variation of circulating IL-6 and glucocorticoid levels (262). Increased endogenous nocturnal secretion of cortisol could alleviate these morning symptoms, but this is not the case in most patients with rheumatoid arthritis (263-270). To clarify this issue, several studies have been carried out reporting that the optimal time for delivery of glucocorticoid treatment would be during the night in order to target the effects of nocturnal proinflammatory stimuli (271-274).

PSYCHOLOGICAL WELL-BEING

The immune system also plays a role in maintaining overall psychological well-being. Chronic stress and its impact on the immune system can increase susceptibility to mental health problems such as anxiety and depression. Conversely, psychological well-being can improve the human body’s immune responses, enhance resistance to diseases (including infectious diseases), and improve mental health (275,276). Changes in psychological status of patients with Alzheimer’s induced significant differences in their immune response (277).

In addition, long-term practice of meditation was shown to decrease stress reactivity and exert a favorable therapeutic effect in chronic inflammatory conditions characterized by neurogenic inflammation (278), while joyful activities such as singing were able to boost the immune response in cancer patients and family members (279).

ADRENAL MEDULLA

The chromaffin cells of the adrenal medulla play a role in stress response by secreting catecholamines and various biologically active peptides (238). As the stress response starts, rapidly augmented secretion of norepinephrine and epinephrine is initiated, followed by activation of the HPA axis and increased release of CRH and ACTH and secretion of glucocorticoids (280). CRH and norepinephrine stimulate the secretion of each other through CRH-R1 and α1-noradrenergic receptors, respectively (281).

Cytokines TNF-α, IL-1, and IFN-γ act directly on chromaffin cells (282-285). It has also been demonstrated that cytokines regulate the secretion of various peptides that are co-secreted with catecholamines, such as vasoactive intestinal peptide (VIP), galanin and secretogranin II, enkephalin and neuropeptide Y (283,285). IL-6 directly modulates the secretion of catecholamines and neuropeptides by chromaffin cells and therefore influences the adrenal stress response. It has been hypothesized that medullary peptides may serve as paracrine modulators of glucocorticoid production (286). It has also been shown that IL-6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells, a finding which has finally confirmed the relations between IL-6 and catecholamine secretion (287). Furthermore, IL-10 is a critical target downstream of epinephrine and norepinephrine which limits inflammation (288). On the other hand, norepinephrine may act directly on macrophages and dendritic cells to suppress inflammatory cytokine secretion through primarily the β2-adrenergic receptors that are expressed by both innate and adaptive immune cells (289,290).

Although scientific advances of the last decade have shed light on the previously unrecognized major role of the catecholamines epinephrine and norepinephrine in controlling the immune system, much remains to be discovered, and further revelations will reveal new therapeutic targets in the management of inflammation.

ACKNOWLEDGEMENTS

This chapter is an update of a previous chapter and the authors would like to thank Professor Gregory Kaltsas, an author of the prior chapter.

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Diabetes and Hypoglycemia in Children and Adolescence

Type 2 Diabetes In Children and Adolescents – A Focus On Diagnosis and Treatment

ABSTRACT

Nearly three decades have passed since the first publications on type 2 diabetes (T2D) in children and adolescents, and it is now well established as a global problem. As the prevalence of obesity continues to increase within the general population, diagnosing T2D in adolescents presents a multifaceted challenge. In this chapter, we shall delve into the epidemiological aspects, as well as the distinct characteristics inherent to various types of diabetes. Cohort studies with long-term follow-ups have illuminated our understanding of the alarming incidence of complications in adolescents diagnosed with T2D. Recent approvals of novel pharmaceutical interventions for teenagers have ushered in a new era of hope. Hopefully, in the forthcoming decade, we anticipate a decline in the prevalence of these complications. However, in the interim, a proactive and assertive approach remains essential for addressing the complications associated with T2D in adolescents.

SHORT HISTORY

Youth-onset type 2 diabetes (T2D) was initially described in Pima Indian children and adolescents (1). This tribe, also known as “the pathfinders,” has a notable history of obesity and early-onset diabetes. In the mid-nineties, studies from various clinics in the United States reported cases of T2D in children from diverse ethnic backgrounds, primarily non-Hispanic Blacks and Hispanics (2-4). While these reports were initially met with suspicion, it soon became clear that a new disease had emerged among children. Subsequently, similar reports emerged from various parts of the world. In 2000, the SEARCH for Diabetes Study was launched in the US. This prospective nationwide, multi-center study aimed at understanding the epidemiology of both type 1 diabetes (T1D) and T2D in children. A few years later, in 2004, the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study was initiated. The TODAY study was a prospective, randomized treatment trial, designed to explore treatment regimens and the clinical course of T2D in youth. Registries from Germany, Diabetes Prospective Follow-up Registry (DPV) (5), Hong Kong (6), China (7), India (Registry of People with Diabetes with Youth Age at Onset (YDR)) (8) and Israel (9), contributed to the current knowledge. Importantly, while the US is leading in the scope of the T2D epidemic, similar trends have been observed worldwide.

EPIDEMIOLOGY

Epidemiology in the USA

According to the most recent data from the SEARCH study published in 2023, the adjusted incidence of T2DM among children and adolescents nearly doubled, from 9.0 to 17.9 cases per 100,000 persons per year, from 2002-03 to 2017-18 (10).

EPIDEMIOLOGY ACCORDING TO ETHNICITY

The incidence of T2D varies largely based on ethnicity, the highest incidence of T2D, 50.1 per 100,000 children age 10-20 years, was recorded in non-Hispanic Blacks, followed by Pima Indians (46), Hispanics (25.8), and Asian/Pacific Islanders (16.6). Among non-Hispanic Whites, the incidence is only 5.5 per 100,000. An annual increase was observed in all groups, but the highest increase was observed in Asian/Pacific Islanders (8.92%), followed by 7.17% in Hispanics and 5.99% in non-Hispanic Blacks, compared to 1.83% in non-Hispanic Whites.

EPIDEMIOLOGY ACCORDING TO GENDER AND AGE

The current incidence in females is 21.6 per 100,000 and in males 14.2; the temporal increase was similar in both genders. The incidence increases with age. One of the most remarkable findings in the SEARCH study is that among individuals 15-19 years of age, the incidence of T2D in 2017-2018 exceeded that of T1D (19.7 vs. 14.6 per 100,000). This is the first time the incidence of T2D surpassed that of T1D among youth, sounding alarming for other countries, which may have a lower incidence than the USA, but are showing steady increases.

Epidemiology in Europe

Although the overall incidence of T2D in Europe is much lower than in the US, a similar change over time has been reported. In Germany, a three-fold increase in the prevalence of T2D was reported for 10- to 19-year-olds between 2002 and 2020 (3.4 to 10.8 per 100,000) (5). Similar to the US, the estimated standardized prevalence of T2D was 1.4 times higher among girls (12.8) than boys (9.0). In the UK, the number of children registered as having T2D and being treated in pediatric diabetes units has risen by more than 50% in the past five years (11).

Epidemiology in Asia

Data retrieved from the Hong Kong Childhood Diabetes Registry revealed a threefold increase in T2D in children, from 1.27 per 100,000 in 1997-2007 to 3.42 per 100,000 in 2008-2018 (6). Data from China demonstrated an average annual increase of 26.6% in youth aged 10-19 years (12). There was no statistically significant difference in incidence between boys and girls. However, the risk of T2D was 1.49 times higher in urban areas than in rural areas.

RISK FACTORS FOR T2D IN CHILDREN

The increased risk of developing T2D in children is associated with genetics, an obesogenic environment, and obesity, along with the interactions among these factors (Figure 1). This visual representation emphasizes the multifaceted nature of risk factors for T2D in children and adolescents, illustrating the intricate connections between genetics, environmental factors, obesity, and the eventual development of T2D.

Figure 1. Risk Factors for T2D in Children and Adolescents. Risk factors for T2D in children and adolescents can be visualized as a series of interconnected circles, illustrating the complex interplay of various influences:
Environment (Outer Circle): The outermost circle represents the broader environmental factors that encompass everything from the intrauterine environment to residential neighborhood.
Genetics (Second Circle): Within the environment circle, genetics forms an important inner circle. It signifies the genetic predisposition that some individuals may have towards developing obesity and, consequently, T2D.
Obesity (Third Circle): Nested within the genetics circle is the obesity circle. It is influenced by multiple factors such as immigration, sedentary lifestyle, diet, microbiome, medication use, depression, and abuse. Of note, not everyone exposed to the obesogenic environment will become obese, indicating the influence of other factors.
Type 2 Diabetes (Innermost Circle): The innermost circle represents the development of T2D. Importantly, not all obese individuals will go on to develop early-onset T2D, highlighting the complexity of these interrelated risk factors.

Obesity

Severe obesity stands out as one of the most significant risk factors for youth-onset of T2D. Obesity is closely linked to the worsening of insulin resistance, a primary feature of the pathophysiology of T2D, along with progressive β-cell and α-cell dysfunction (13). In a recent meta-analysis comprising data from 30 studies involving 4688 children and adolescents, the prevalence of obesity at the time of T2D diagnosis was found to be 77 % (95% CI, 71%-83%) (14). Male participants exhibited higher odds of obesity than their female counterparts (odds ratio, 2.10; 95% CI, 1.3-3.3). These findings were sustained after several sensitivity analyses that excluded studies with uncertain or unspecified T2D diagnostic criteria, studies involving individuals with positive pancreatic autoantibodies, individuals presenting with weight loss, or those with genetically proven monogenic diabetes. While acknowledging the limitations of using BMI-based measures as a surrogate for obesity and considering the retrospective nature of the included studies, these data suggest that approximately 20-25% of adolescents with diabetes but without obesity may still have T2D. Furthermore, the definition of obesity needs to consider body composition variations among certain populations, such as East Asian and South Asian populations.

Family History of T2D

Most adolescents with T2D have a family member with the disease. In the TODAY study, 59.6% of adolescents with T2D had a first-degree family member with a history of diabetes, and 89% had a grandparent affected with diabetes (15). This increased risk of T2D associated with family history reflects genetic influences, the impact of the environment, and the intrauterine environment.

Maternal Obesity and Maternal T2D During Pregnancy

Exposure to maternal obesity has been linked to an increased risk of childhood obesity in offspring (16) and 2.8-fold higher odds of T2D. Exposure to maternal gestational diabetes was associated with 5.7-fold higher odds of T2D (17). Moreover, individuals exposed to maternal diabetes during pregnancy were diagnosed with diabetes at a younger age and exhibited worse β-cell function. Exposure in utero to maternal diabetes and obesity accounted for 47% of the T2D risk in this population (17,18), and, importantly, it sets up a vicious cycle for future generations. Suggested mechanisms affecting the developmental programming of offspring towards T2D include epigenetic modifications, alterations in stem cell differentiation, variation in the metabolome and microbiome, and immune dysregulation (19). Of note, assistedreproductive technology was not found to be a risk factor for early-onset T2D (20).

Pregnancies complicated by pre-existing diabetes are associated with extreme birth weights. In a large study, small for gestational age (SGA) babies, defined as below the 10th percentile for birth weight, were reported in 14.1% of pregnancies of women with T2D, while 26.2% had newborns with a large birth weight, defined as above the 90th percentile (21). Both SGA and high birth weight are associated with an increased risk of a history of early-onset T2D among adults. Of note, a lower birthweight was associated with an increased risk of developing T2D independently of adult BMI and the genetic risk of T2D (22).

Pregnancies complicated by pre-existing diabetes are associated with preterm delivery and birth weight extremes. In a national cohort study involving over 4 million singletons born in Sweden from 1973 to 2014, preterm birth (occurring before 37 weeks) was associated with a 1.3-fold increased risk of developing T2D before age 18 years (23). Notably, the association between preterm birth and T2D was significantly stronger among females (23).

Mental Health and Treatment

The association between psychiatric disorders in adolescents and T2D is bidirectional, complex, and not well studied in the pediatric population (24). On the one hand, youth with chronic illnesses such as obesity are more likely to develop depression, depressive symptoms, and anxiety than those without chronic illnesses. On the other hand, youth who have mental health morbidities are at increased risk for isolation, sedentary lifestyle, weight gain, and the development of T2D. Furthermore, some psychotropic medications, particularly atypical antipsychotics, are associated with weight gain and increased risk for T2D in adults. These associations have not been systematically studied in youth, but clinical experience suggests a contribution.

Immigration

Globally, immigration is on the rise. Studies conducted among adults have indicated that immigrants, when compared to non-immigrants, exhibit higher rates of obesity, insulin-resistance, and hypertension. In a recent review (25) focusing on European countries, non-European migrant children face a greater risk of being overweight or obese compared to their native counterparts; the prevalence of obesity in migrant and native children ranged from 1.2 to 15.4% and from 0.6 to 11.6%, respectively. The increased rates of overweight and obesity among migrating children and adolescents render them more susceptible to developing T2D and other metabolic abnormalities. An Israeli study found that among adolescents of Ethiopian origin, the prevalence of overweight and obesity increased two-and-a-half fold and fourfold in males and females, respectively, during the study period, compared to a 1.5-fold increase among native Israeli-bornmales and females (26).

Socioeconomic Status

A discernible disparity exists in the socioeconomic status (SES) of children and adolescents with T2D. In developed countries, such as the United States, low SES is a recognized risk factor for the development of obesity and T2D. This is attributed to factors such as limited access to healthy foods and reduced physical inactivity. However, in developing countries, higher SES groups tend to have greater levels of physical inactivity and consume higher quantities of fat, salt, and processed foods compared to their lower SES counterparts (Figure 2). When data from 384 youth with T2D who completed a baseline research visit as part of the SEARCH study and 227 youth with T2D from the Registry of People with Diabetes with Youth Age at Onset (YDR) in India who completed a baseline visit were compared, only 23.6% of SEARCH youth belonged to a high SES group, in contrast to 88.5% of YDR youths (P < .001) (8).

Figure 2. Global Disparities in Type 2 Diabetes Risk Factors. This illustrative figure offers insights into the diversity in risk factors for T2D among children across the world. It highlights how these risk factors vary across different regions and populations.
Gender Disparities (Upper Part of Figure): In many Western countries, the risk of developing T2D in children is higher in girls compared to boys. However, this pattern is not consistent globally. In other parts of the world, an equal or even higher risk of T2D has been documented in males. The upper portion of the figure visually represents these variations in risk, depicting the female-to-male ratios in different regions.
Socioeconomic Status (SES) Variation: (Lower Part of Figure) SES plays a significant role in T2D risk. In Western countries, lower SES is associated with an increased risk of T2D in children. Conversely, in developing countries, a contrary trend is observed, with a higher risk of T2D observed in populations with higher SES. These complex socioeconomic variations are highlighted in the figure, showcasing how T2D risk is influenced by economic factors on a global scale.

Data from 747 children and youths with T2D under 19 years of age, collected between 2009 and 2016 (from the population-based National Pediatric Diabetes Audit, covering over >95% of diabetes cases in England and Wales), revealed that under half of those with T2D reside in the most disadvantaged areas of England and Wales, and 41% fall into the most disadvantaged SES quintile (27). In contrast, in China, in Zhejiang, one of the most economically prosperous coastal provinces marked by industrialization and urbanization, changes in diet and decreased physical activity have led to a higher incidence of pediatric obesity. The mean annual incidence was 2.32 per 100,000 person-years in urban area compared with 1.44 in urban and rural area (12).

COVID-19 Pandemic

In a study conducted in the United States, the incidence of T2DM at 24 diabetes centers during the first year of the COVID-19 pandemic was examined (28). The average number of new diagnoses per year in the two years before the pandemic was 825 but rose to 1463 during the first pandemic year. This increase of 77% is significantly higher than the 5% expected annual increase in incidence observed in the two previous years. Similarly, a retrospective cross-sectional review of youths (age ≤ 21) diagnosed with T2D during the COVID-19 pandemic (from May 1, 2020, to April 30, 2021) and the five years preceding it (from May 1, 2015, to April 30, 2020) at a tertiary diabetes center revealed an increase of 293% (29). Similar trends were reported from Germany (30). Data on T2D in adolescents during 2 years of the COVID-19 pandemic (2020–2021) were compared with the control period 2011–2019 in children aged 6 to <18 years were obtained from the DPV (German Diabetes Prospective Follow-up) Registry. The incidence of youth-onset T2D increased from 0.75 per 100,000 patient-years in 2011 to 1.25 per 100,000 in 2019, an annual increase of 6.8%. However, in 2021, the observed incidence was 1.95, significantly higher than expected. Of note, the observed incidence was significantly higher in boys (2.16), leading to a reversal of the sex ratio of pediatric T2D incidence (30). The surge in T2D incidence during the pandemic can be attributed to several factors. First, there was an increase in obesity among young people during the COVID-19 pandemic, accompanied by increased consumption of processed foods and reduced physical activity, both of which contribute to the risk of developing T2D (31). In addition, there was increased psychosocial stress, which is emerging as an important contributor to the risk of T2D. Furthermore, viral mediated non-autoimmune β-cell destruction, resulting in reduced β-cell function in predisposed adolescents, has been suggested as a contributing factor (28).

Genetics

The genetics of T2D in children and adolescents have largely remained unexplored. ProDiGY is a multiethnic collaboration encompassing three studies (TODAY, SEARCH, and T2D-GENES) involving 3,006 youth subjects with T2D, diagnosed at a mean age of 15.1±2.9 years, along with 6,061 diabetes-free adult control subjects. Association analyses were conducted on approximately 10 million imputed variants, employing a generalized linear mixed model incorporating a genetic relationship matrix to account for population structure. The analysis was further adjusted for sex. This comprehensive study identified seven genome-wide significant loci that included a novel locus in PHF2, along with TCF7L2, MC4R, CDC123, KCNQ1, IGF2BP2, and SLC16A11. A secondary analysis involving 856 diabetes-free youth subjects uncovered an additional locus in CPEB2, and consistent directional effects for diabetes risk were observed (32).

CLINICAL PRESENTATION

Diabetes Related Signs

The classical signs at the onset of diabetes - polydipsia and polyuria - are observed in about two-thirds of youth at the diagnosis of T2D. Only about one-third are diagnosed through routine screening of asymptomatic youth with obesity. Furthermore, the prevalence of diabetic ketoacidosis (DKA) among youth with T2D in the SEARCH study and the YDR in India was 5.5% and 6.6%, respectively. Importantly, the incidence of DKA at the onset of T2D was higher during the SARS-CoV-2 pandemic (33). Hyperglycemic hyperosmolar state is present at diagnosis in 2% of youth with T2D (34).

Seasonal Diagnosis of T2D

There is significant seasonal variation in the onset of T2D in children and young people in the United States; diagnoses increased in August. Possible explanations for an August peak in diagnoses include weight gain during the summer vacation and an increase in physical exams for school athletic programs that may detect asymptomatic hyperglycemia. The greater proportion of diagnoses made during routine health visits rather than following symptoms is consistent with this latter possibility. Similar seasonal variations in incidence are beginning to emerge in other countries.

Age of Onset of T2D

The incidence of T2D rises gradually during puberty, primarily due to the physiological insulin resistance characteristic of this period. However, illustrating the heterogeneity of T2D in children and adolescents, there was a significant difference between the mean age of onset of youth in the United States (Pediatric Diabetes Consortium, PDC) databases who were diagnosed at the mean age of 12 years compared to the mean age in Germany of 13 years (35).

PREPUBERTAL T2D

With the worldwide epidemic of childhood obesity on the rise, there are increasing reports of T2D occurring in prepubertal children. Initially, there were case reports on prepubertal children with T2D, including cases from Nigeria (36), and a 5-year-old Indigenous girl from Australia (37). Lately, several reports have described the onset of T2D in prepubertal children. A total of 42 children ≤ 10 years with T2DM were reported from Alabama (38), 12 from Australia (39), 35 prepubertal children from Houston, Texas (40), from San Antonio, Texas (41) and India (42). The common denominator of all reports of prepubertal children is extreme obesity, with a disproportionate impact on females and ethnic minorities as shown in Table 1. Additionally, there is a high prevalence of comorbidities.

Table 1. Characteristics of Prepubertal Children Diagnosed with T2D
	San Antonio, Texas N=20	Alabama, US N=42	Australia N=12	Houston, Texas. N=35	Chennai, Tamil Nadu India N=4
Age, years Range	8.1 4-9		7-10	10.6± 2.5
5-8	10	8
9-10	10	34
Ethnicity	Hispanic 80%	African American 88(%) Caucasian 12%	Aboriginal Australians 92 Maori 8	Hispanic 71 Black 23 Non Hispanic White 3%	India
Females (%)	75	88	75	51	100
BMI Z score	2.72 (1.7-5.0)	2.5 ± 0.4	2.38± 0.64	2.4±0.4	>97%ile
Hypertension (%)	NA	21	25	14
Dyslipidemia	NA	58	58%	100
NAFLD	NA			20
Microalbuminuria	NA		16.6	5

These reports suggest that there are unique features and pathophysiologic drivers distinct from the influence of pubertal hormones, which are often implicated in the mechanism underlying T2D in older children and adolescents. Furthermore, the emergence of T2D in prepubertal children and the high prevalence of comorbidities among them serve as a warning for an impending public health challenge.

Sex

In the Western world, youth-onset T2D is almost twice as common in girls as in boys, whereas Asian countries report no differences in incidence by sex. For T2D, SEARCH had a higher proportion of females, while studies from China show no difference between females and males, and there are reports of increased prevalence in males in the Middle East; in the UAE (43), the female to male ratio was 0.5:1, in Iraq (44) 0.6:1, and Kuwait (45) 0.8:1 (Figure 2). This may reflect the higher prevalence of obesity in males in these countries (46). Indeed, in the UAE, among children aged 11 to 14 years, the prevalence of obesity was reported as 24.3% and extreme obesity (BMI ≥99^th percentile) as 5.7%; the rate of extreme obesity was 9.6-fold higher in boys than girls (47). In Kuwait, 41.4% of boys were classified as obese, compared to 28.9% of girls of the same age (46).

DIAGNOSIS

Early diagnosis of T2D is likely to bring several benefits, enabling prompt multifactorial treatment and management of cardiovascular risk factors. Considering the increasing prevalence of T2D among children and adolescents, clinical symptoms have become less distinct, and age, sex, and weight are no longer definitive criteria for a clear diagnosis. Therefore, the most important diagnostic feature is the presence or absence of pancreatic autoantibodies in any child diagnosed with hyperglycemia; positive antibodies indicate T1D even if the child is overweight or has a family history of T2D. If the antibodies are negative, a genetic test for monogenic diabetes (previously referred to as Maturity-Onset Diabetes of the Young (MODY) should be considered if available.

TREATMENT

Initial treatment for T2D includes a focus on lifestyle changes, including healthy diet, increasing regular physical activity, achieving weight loss, and receiving emotional support. While lifestyle change is recommended as a practical approach,there is limited evidence that lifestyle intervention alone has a sustained impact on glycemic control in youth with T2D (48).

The Impact of Lifestyle Changes

The impact of lifestyle intervention, as determined by changes in diet and cardiovascular fitness, on glycemic control in youth with T2D was assessed in the TODAY clinical trial cohort spanning 15 centers across the United States. The study included 699 youth aged 10 to 17 years with T2D for less than 2 years. Those participants randomly assigned to the lifestyle intervention participated in a family-based behavioral program aimed at promoting weight loss. Each family was assigned a dedicated leader responsible for guiding them through their journey of physical activity and nutrition improvement. The intervention was structured across three stages, initially there were weekly meetings for 6 to 8 months, focusing on physical activity, setting individual calorie intake goals, self-monitoring, and problem-solving. In the subsequent stage, biweekly meetings were held for 12 to 16 months. Lastly, meetings were held monthly for 24 to 28 months, concentrating on maintaining a healthy lifestyle. Dietary data were collected through an interviewer-administered food frequency questionnaire, and cardiovascular fitness was assessed using a submaximal cycle ergometer test at baseline, 6 months, and 24 months (49). At 6 months, approximately 25% of females and 33% of males improved cardiovascular fitness. The authors concluded that a minority of youth improved fitness and/or diet over time, although those who did showed a beneficial impact on glycemic outcomes. However, the positive lifestyle behavior changes did not persist after 24 months, providing a sobering perspective on the challenges of implementing lasting lifestyle changes in this population.

Pharmacotherapy

The beginning of the third decade of the 21st century will be marked by the introduction of novel medications for children with T2D. There are several groups of medications for T2D, biguanides, sulfonylureas, GLP1 receptor agonists, SGLT2 inhibitors, dipeptidyl peptidase inhibitors, and combinations of the dual incretin analogs and the new triple G agents (dual incretin and glucagon). However, only biguanides, GLP1 receptor agonists, and SGLT2 inhibitors are currently FDA-approved for children. Doses and modes of administration are presented in Table 2.

Table 2. FDA-Approved Medications for Treating Type 2 Diabetes in Children Aged 10 Years and Older
Biguanide	Metformin	Glucophage	p.o daily	850, 1000. up to 3000 day
GLP receptor agonist	Liraglutide	Victoza	s.c. daily	0.6mg 1.2mg 1.8mg
	Exenatide	Bydureon	s.c weekly	2 mg
	Dulaglutide	Trulicity	s.c weekly	0.75mg/0.5mL 1.5mg/0.5mL 3mg/0.5mL 4.5mg/0.5mL
SGLT2 inhibitor	Empagliflozin	Jardiance	p.o daily	10 mg 25 mg
SGLT2 inhibitor	Dapagliflozin	Forxiga	p.o daily	5mg 10 mg

Five randomized control studies assessed the impact of these drugs in children with T2D. The inclusion criteria were consistent across all studies and included an age above 10 years, a BMI above the 85^th percentile, and minor variations in HbA1c levels (between 7.0 and 11.0%, or 6.5% and 10%). The characteristics of the participants and outcomes are detailed in Table 3. Figure 3 depicts HbA1c results vs. placebo for each medication, and Figure 4 depicts the percent of individuals with HbA1c less than 7% at the end of each study.

Table 3. Effects of Various Medications on Glycemic and Anthropometric Parameters in Adolescents with Type 2 Diabetes After 26 Weeks of Treatment
	Liraglutide		Exenatide		Dulaglutide 0.75 mg		Dulaglutide 1.5 mg		Empagliflozin		Dapagliflozin		Linagliptin		Sitagliptin
	Tx	Pl	Tx	Pl	Tx	PL	Tx	PL	Tx	PL	Tx	PL	Tx	Pl	Tx	Pl
Number	66	68	59	24	52	51	52	51	52	5	39	33	53	53	95	95
Age (years)	14.6	14.6	15	16	14.7	14.2	14.7	14.2	14.6	14.4	16.1	14.4	14.6	14.4	14.3	13.7
Baseline HbA1c )%(	7.9	7.7	8.1	8.1	7.9	8.1	8.2	8.1	8.0	8.05	7.95	7.85	8.05	8.05	7.4	7.6
HbA1c change	-0.64	0.46	-0.36	0.49	-0.6	0.6	-0.9	0.6	-0.17	0.68	-0.25	0.5	0.33	0.68	-0.01	0.18
HbA1c ETD	1.06		0.85		1.2		1.5		0.84		0.75		0.35		0.19
% HbA1c <7%	63.7	36.5	48	35.1	55	14	48	14	34.6	24.5	25	4	26.5	24.5	49.5	36.8
Baseline BMI (kg/m²)	34.6	33.3	36.9	35.4	33.6	34.3	34.3	34.3	35.5	36.1	31.3	33.6	36.5	36.1	0.0	-0.7
BMI ETD	-0.25	-0.21	ND	ND	-0.2	0	-0.1	0	ND	ND	-0.08	-0.11	ND	ND	-0.7
Weight change (kg)	-2.3	-0.99	-0.59	0.63	0.3	0.1	0.2	0.1	-0.79	-0.04	ND	ND	1.42	-0.04	-1.1	0.06
Weight ETD	-1.3		-1.2		0.2		0.1		-0.75				1.46		1.04
Systolic BP ETD	0.03		-2.9		3.8		2.5		-1.42		1.9		0.91
Diastolic BP ETD	-1.08		ND		-0.8		-2.6		0.02		-0.5		1.5

Pl=placebo, Tx= treatment, ETD = estimated treatment difference, ND= no data, placebo group for Empagliflozin and Linagliptin was the same.

Figure 3. Change in HbA1c percentage points in adolescents with T2D under medication treatment compared with untreated control group. The differences in HbA1c percentage points from the baseline for adolescents diagnosed with T2D who received medication treatment (depicted in black) compared to control groups (shown in white with data points). In the medication-treated group, there is a significant decrease in HbA1c levels, denoted by the downward trend in the black bars. Conversely, in the control groups of all the studies, HbA1c levels increased, as indicated by the upward trend in the white bars with data points.

Figure 4. HbA1c Levels below 7% in adolescents with T2D. The percentage of adolescents who achieved HbA1c levels below 7% at the end of the follow-up period. A comparison is drawn between those treated with medication (depicted in black) and the control group (depicted in white).

METFORMIN

Metformin is a biguanide antihyperglycemic used in conjunction with diet and exercise to control glycemia. Metformin is considered the preferred first-line agent for treating T2D in pediatric patients 10 or older (50). Its onset of action is around 1.5 hours, and its total duration of action is 16-20 hours. Metformin exerts its glucose-lowering effect through various mechanisms, including inhibiting hepatic gluconeogenesis, increasing glucose uptake in skeletal muscles, reducing adipogenesis, and activating brown adipose tissue, leading to enhanced thermogenesis. Additionally, metformin decreases the absorption of glucose from the intestine and downregulates inflammation.

Although it is the first line of treatment for children and adolescents with T2D, prospective, randomized, controlled, long-term studies are limited. In a randomized control study involving 82 children and adolescents, 42 received metformin at doses up to 2000 mg/day, and 40 received placebo (51). After 16 weeks, mean HbA1c levels were lower in the metformin group compared to the placebo (7.5% and 8.6%, respectively). The metformin group also experienced a mean weight decrease of -1.5 kg compared with a mean increase of 0.9 kg in the placebo group. The mean BMI changed by -0.5 units in the treatment group vs. -0.4 units in the placebo group. In another study, metformin (500–1000 mg twice daily) was compared to glimepiride, a sulfonylurea (1–8 mg once daily), for 24 weeks (52). Both metformin (-0.71%) and glimepiride (-0.54%) groups showed significant reductions from baseline HbA1c levels. A total of 48.1% of metformin-treated and 42.4% of glimepride-treated participants achieved HbA1c levels below 7.0% at week 24. While the incidence of hypoglycemia was similar in both groups, metformin resulted in less weight gain compared to glimepiride. In a large observational study (53), 927 youth aged 13.7±2.0 years old, who had been diagnosed with T2D for a median of 2 months and had a baseline HbA1c of 7.7±2.2%, were treated with metformin. After a median of 71 days, the mean HbA1c change was -1.33, and the mean weight change -0.43 kg (53,54).

Metformin-related side effects are gastrointestinal, including diarrhea, abdominal pain, bloating, nausea, and decreased appetite, which occur in about 50% of users. Gastrointestinal side effects are usually transient and improve over time. Another potential side effect of metformin is vitamin B12 deficiency, and regular monitoring is indicated, though no cases of vitamin B12 deficiency were identified in the TODAY study. Lactic acidosis is a concerning side effect associated with metformin use in adults. It occurs in the presence of hypoperfusion or hypoxemia; however, it has rarely been reported in children and adolescents.

GLUCAGON-LIKE-PEPTIDE 1 (GLP1)

Glucagon-like peptide-1 (GLP-1) is a peptide hormone primarily produced by intestinal enteroendocrine cells at low basal levels that rapidly increases within minutes of food consumption. GLP-1 plays a vital role in regulating meal-related glycemic excursions by enhancing insulin secretion and inhibiting glucagon secretion. Additionally, GLP-1 slows gastric emptying and reduces food intake through a central effect on appetite, which contributes to weight loss. However, GLP-1 has a short half-life of approximately 2 minutes, as it is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). To enhance GLP-1 activity, medications such as DPP-4 inhibitors and GLP-1 receptor agonists have been developed.

The story of the development of GLP1 agonists is truly fascinating. It begins with the venomous Gila monster (Heloderma suspectum), native to New Mexico and Arizona. H. suspectum, a long-lived and reclusive species, spends a significant portion of its life underground. Although its venomous bite causes pain and weakness, it is rarely fatal to adult humans. In 1990, endocrinologist Dr. John Eng, while analyzing the venom to identify new hormones, identified a peptide named exendin-4. He discovered that exendin-4 had a remarkable ability to stimulate the synthesis and release of insulin from β-cells in the pancreas. Interestingly, exendin-4 closely resembled GLP-1. However, while GLP-1 remains active for about two minutes, the effect of exendin-4 persists for several hours. Preclinical studies demonstrated that a single daily injection of exendin-4 normalized blood glucose concentrations in mice with diabetes. Following extensive clinical testing, exenatide, an analog of exendin-4, was found to be safe and effective, leading to its FDA approval for adults in 2005.

Of note, in a 2-year rat carcinogenicity study involving prolonged-release exenatide, an increased incidence of thyroid adenoma and C-cell carcinoma was observed at doses ≥2-fold the human systemic exposure. The clinical relevance of these adverse findings is currently unknown. However, GLP-1 analogs are contraindicated in patients with a personal or family history of medullary thyroid carcinoma (MTC) or in patients with multiple endocrine neoplasia type 2 (MEN 2), as well as in patients with a serious hypersensitivity reaction. Patients should be counseled about the potential risk of MTC associated with the GLP1 receptor agonists, and it is important to inform them about the symptoms that may indicate thyroid tumors, such as a neck mass, dysphagia, dyspnea, and persistent hoarseness. Routine monitoring of serum calcitonin levels or use of thyroid ultrasound for the detection of MTC is of uncertain value.

Side effects include serious hypersensitivity reactions, including anaphylactic reactions and angioedema. Additionally, acute pancreatitis, including fatal and nonfatal hemorrhagic or necrotizing pancreatitis, and the acute onset of gallbladder disease, such as cholelithiasis or cholecystitis, have been reported in GLP-1 receptor agonists trials and post-marketing surveys (55).

Liraglutide-Victoza®

Children aged 10 to less than 17 years with T2D were randomly assigned 1:1 to receive subcutaneous liraglutide (up to 1.8 mg per day) or placebo for a 26-week double-blind period, followed by a 26-week open-label extension (56). At the 26-week visit, the mean HbA1c had decreased by 0.64 percentage points with liraglutide and increased by 0.42 percentage points with placebo, resulting in an estimated treatment difference of -1.06 percentage points. By 52 weeks, the difference had increased to -1.30 percentage points (Table 3). In the liraglutide group, 63.7% of patients achieved HbA1c levels of less than 7.0%, compared with 36.5% in the placebo group. On the other hand, there was no statistical benefit of liraglutide over placebo in lowering BMI or blood pressure (Table 3). The overall rate of adverse events, including gastrointestinal adverse events, was higher among patients receiving liraglutide. The U.S. Food and Drug Administration approved liraglutide injection for the treatment of pediatric patients 10 years of age or older with T2D. Liraglutide was the first non-insulin drug approved to treat T2D in pediatric patients since metformin was approved for pediatric use in 2000.

Mode of administration: The recommended initial dose is 0.6 mg subcutaneously once daily for the first week, followed by an increase to 1.2 mg once daily. It’s important to note that the initial dose of 0.6 mg once daily is primarily intended to mitigate gastrointestinal adverse effects and does not provide glycemic control. If adequate glycemic control is not achieved, the dose can be further increased to 1.8 mg once daily.

Exenatide-Bydureon

The effectiveness of once-weekly exenatide 2 mg (Bydureon AstraZeneca) in youth with suboptimally controlled T2D was evaluated (57). At 24 weeks, exenatide demonstrated superiority over the placebo in reducing HbA1c levels (a decrease of -0.36% with exenatide compared to +0.49% with placebo), resulting in a significant between-group difference of 0.85 percentage points. Notably, no major hypoglycemic events were reported. and the most frequently reported adverse events included gastrointestinal symptoms such as nausea, diarrhea, and vomiting, along with injection site reactions such as pruritus, erythema, and nodules. Clinically meaningful differences were not observed in change from baseline in BMI Z score, body weight, or waist circumference, nor were there significant differences in blood pressure. The prolonged-release suspension of exenatide administered via injection with a pre-filled pen has received approval from the European Union and the FDA for the treatment of T2DM in children and adolescents aged 10 years and older.

Mode of administration: Bydureon (2 mg per dose) should be administered once every 7 days. The dose can be administered at any time of day, with or without meals. Bydureon is not recommended as first-line therapy for patients with inadequate glycemic control on diet and exercise because of the uncertain relevance of the rat thyroid C-cell tumor findings to humans.

Dulaglutide-Trulicity

Dulaglutide (Trulicity) is a GLP-1 receptor agonist administered via subcutaneous injections once a week. In a randomized, double-blind, phase 3 trial involving youth with inadequately controlled T2DM, the efficacy and safety of dulaglutide treatment in reducing HbA1c levels at 26 weeks were assessed (58). Remarkably, there was 99% adherence to treatment in this trial. Treatment resulted in a significant reduction in HbA1c relative to placebo; at week 26, HbA1c decreased by 0.8 percentage points but increased by 0.6 percentage points in the placebo group (1.4 percentage point difference). Gastrointestinal symptoms were among the most common adverse events, but they were primarily mild and were most likely to occur soon after the initiation of therapy. There were no clinically meaningful differences in the incidence or annual rate of hypoglycemia between the dulaglutide groups and the placebo group. Body weight did not decrease significantly following treatment.

Mode of administration: The recommended initiating dose of Trulicity is 0.75 mg once weekly, administered subcutaneously. The dose may be increased to 1.5 mg once a week for additional glycemic control. Trulicity can be administered any time of day, with or without meals, and should be injected subcutaneously in the abdomen, thigh, or upper arm. The day of weekly administration can be changed, if necessary, as long as the last dose was administered 3 days (72 hours) or more before. Trulicity and insulin should not be mixed in the same syringe and must be administered as two separate injections at two different injection sites.

SODIUM-GLUCOSE TRANSPORT PROTEIN 2 - SGLT2 INHIBITORS

Sodium-glucose transport protein 2 (SGLT2) inhibitors belong to a class of medications that modulate sodium-glucose transport proteins in the nephron. These inhibitors primarily target the SGLT2 proteins, expressed in the renal proximal convoluted tubules, to reduce the reabsorption of filtered glucose and sodium (59). Apart from their role in controlling glucose concentrations, SGLT2 inhibitors have demonstrated substantial cardiovascular benefits, particularly reducing heart failure events, for adults with T2D. In adults, the FDA-approved indications for SGLT2 inhibitors include the reduction of major adverse cardiovascular events in individuals with T2D and established cardiovascular disease, as well as the reduction of the risk of eGFR decline in patients with chronic kidney disease at risk of progression. The most common reported adverse events associated with SGLT2 inhibitors include female genital mycotic infections, urinary tract infections (including urosepsis and pyelonephritis), as well as nausea and constipation. Most importantly, SGLT2 inhibitors are associated with an almost three-fold increased risk for DKA in adults. The risk for DKA is highest for canagliflozin, followed by empagliflozin and dapagliflozin. Furthermore, individuals may develop euglycemic DKA, i.e., a triad of increased anion gap acidosis, ketosis, and a serum glucose level below 250 mg/dL. The association between SGLT-2 inhibitors and euglycemic DKA appears to be secondary to their noninsulin-dependent glucose clearance, compensatory hyperglucagonemia, and volume depletion. Currently, euglycemic DKA has not been reported among youth with T2D taking SLGT2 inhibitors.

Empagliflozin - Jardiance®

A total of 105 children and adolescents were randomly assigned 1:1 to receive empagliflozin 10 mg or placebo (60). The adjusted mean change in HbA1c from baseline at week 26 was significantly greater in the empagliflozin group (–0·84%). Changes in body weight and blood pressure did not reach statistical significance. Adverse events occurred in 64% of participants in the placebo group and 77% in the empagliflozin group up to week 26, including severe adverse events in 4% of participants in the placebo group and 2% in the empagliflozin group. Mild and moderate hypoglycemia were the most frequently reported adverse events, with higher rates for those on active drug treatment compared with placebo; no severe hypoglycemic events were reported, and there were no episodes of DKA or necrotizing fasciitis associated with empagliflozin treatment. An increased incidence of bone fractures has been reported in adults, leading the American Diabetes Association to recommend avoiding SGLT-2 inhibitors in patients with a risk factor for fractures. Furthermore, SGLT2 inhibitors are not recommended for glycemic lowering in patients with T2D with an eGFR less than 30 mL/min/1.73 m².

Mode of administration: Empagliflozin is an oral medication dosed at either 10 mg daily or 25 mg daily. The recommended dose is 10 mg once daily in the morning, taken with or without food. If tolerated initially, dosing may increase up to 25 mg.

Dapagliflozin - Farxiga®

Participants aged 10–24 years with T2D and HbA1c concentrations ranging from 6.5 to 11% were given oral dapagliflozin 10 mg or placebo during a 24-week double-blind period (61); 39 participants were assigned to dapagliflozin and 33 were assigned to placebo. After 24 weeks, the mean change in HbA1c concentration was −0·25% for dapagliflozin and 0·50% for the placebo group; the between-group difference was −0·75%, which did not reach statistical significance. However, in a sub-analysis, excluding participants with poor compliance, the HbA1c change was -0.51%, while it was 0.62% for the placebo group, resulting in a between group significant difference of 1.13%.

Adverse events occurred in 69% of participants assigned to dapagliflozin and 58% of those assigned to placebo over a 24-week period. Over 52 weeks, adverse events occurred in 74% of participants who received dapagliflozin. Hypoglycemia was noted in 28% of participants assigned to dapagliflozin and in 18% of those assigned to placebo, none were considered serious adverse events. Notably, there were no reported episodes of DKA.

Mode of administration: To improve glycemic control the recommended starting dose is 5 mg once daily, taken in the morning. Increase dose to 10 mg once daily in patients tolerating 5 mg who require additional glycemic control. Dapagliflozin is not recommended for use in patients receiving loop diuretics or who are volume depleted, e.g., due to acute illness (such as gastrointestinal illness).

DIPEPTIDYL PEPTIDASE 4 (DPP 4) INHIBITORS

DPP-4 is a ubiquitous enzyme that degrades GLP-1 and GIP (gastric inhibitory peptide), resulting in a short half-life. By inhibiting the DPP-4 enzyme, DPP-4 inhibitors increase the levels of GLP-1 and GIP. This, in turn, enhances beta-cell insulin secretion, thus reducing postprandial and fasting hyperglycemia (62).

Linagliptin (Tradjenta)

105 children and adolescents were randomly assigned 1:1 to oral linagliptin (5mg daily) or placebo (60). The adjusted mean HbA1c change from baseline at week 26 was 0.33% for linagliptin and 0·68% for the placebo group; the difference was not statistically or clinically significant. None of the adverse events of special interest occurred with DPP-4 inhibitors, such as hypersensitivity reactions, angioedema, angioedema-like events and anaphylaxis, skin lesions, pancreatitis, or hepatic injury, were present. Linagliptin is not approved for adolescents with T2D (60).

Sitagliptin (Januvia)

A total of 190 children and adolescents were randomly assigned 1:1 to oral sitagliptin 100 mg daily or placebo (63). The adjusted mean change in HbA1c from baseline at week 20 was -0.01% for sitagliptin and 0·18% for the placebo group, resulting in a between-group difference of 0.19%, which was not significant. There was no significant difference in the percentage of participants with HbA1c below 7%, with 49.5% in the sitagliptin group and 36.8% in the placebo group. Notably, after 54 weeks, the HbA1c in the sitagliptin group decreased from 7.4% at baseline to 7.1%. Sitagliptin is not approved for adolescents with T2D (50).

ANTI-OBESITY PHARMACOTHERAPY FOR TREATMENT OF PEDIATRIC TYPE 2 DIABETES

As obesity is a significant risk factor for the development of T2D, the American Diabetes Association (ADA) recommends the use of anti-obesity medications as adjuvant therapy for adults with both T2D and overweight/obesity. In adults, adding anti-obesity medications to a diabetes regimen can improve glycemic control, reduce weight, and decrease the use of anti-diabetes medication (64). Semaglutide and tirzepatide have shown promise in reducing weight in adults with obesity and T2DM compared to liraglutide (65). Tirzepatide is a novel dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist. A study to evaluate tirzepatide in pediatric and adolescent participants with T2D, who are inadequately controlled with metformin, or basal Insulin, or both (SURPASS-PEDS) is currently ongoing.

Liraglutide 3.0mg/day

A higher dose of liraglutide of 3.0 mg/day (Saxenda^®) is FDA-approved for obesity in youth aged 12 years and older who weigh ≥ 60kg or have an initial BMI ≥ 30kg/m². The Satiety and Clinical Adipose-Liraglutide Evidence Trial in Adolescents with and without T2D (SCALE-Teens) was a double-blind, placebo-controlled RCT that randomized 251 adolescents to either liraglutide 3.0 mg/day or placebo. However, only two adolescents with T2D were enrolled. Consequently, no conclusive findings can be drawn regarding the impact of high-dose liraglutide on reducing BMI and improving HbA1c in young individuals with T2D. Since almost all adolescents with T2DM have overweight or obesity and could potentially meet the prescribing criteria, it is reasonable to speculate that the results might resemble those observed in adult trials for T2D, potentially leading to reductions in weight and BMI as well as improvements in hyperglycemia.

Semaglutide 2.4mg Weekly

Semaglutide in its once-weekly injectable GLP-1RA form at doses up to 2.4 mg weekly (Wegovy^®) has shown significant weight loss in obese children and has now been approved for treatment of obesity in adolescents aged 12 years and older (66). The mean change in BMI from baseline to week 68 was −16.1% with semaglutide and 0.6% with placebo. Of note, HbA1c decreased 0.4 percentage points in the treatment group compared with 0.1 in the placebo. However, the impact on youth with T2D has not yet been studied.

Semaglutide injection up to 2.0 mg (Ozempic^®) and oral semaglutide up to 14 mg (Rybelsus^®) are approved for adults with T2D, but studies in youth with T2D have not yet been reported.

The answer to this pivotal question hinges on the art of personalized medicine. First, it is crucial to recognize that 50% of youth with T2D can maintain good glycemic control on metformin monotherapy, making this agent still an excellent choice for initial therapy. However, several measures can predict the likelihood of glycemic deterioration, including decreased insulin secretion, impaired insulin processing (elevated proinsulin/insulin ratio), and HbA1c concentration after a few months on monotherapy. Longitudinally, a rising proinsulin and an increase in HbA1c of more than 0.5 percentage points over any 6-month period are predictive of loss of glycemic control on monotherapy (67).

If these parameters suggest a risk for deterioration, the next step is to consider the therapeutic objective(s). Is it primarily elevated HbA1c? If so, the best approach may involve selecting a medication known for its efficacy in reducing HbA1c. Is obesity the central concern for the patient? In such cases, exploring pharmaceutical interventions tailored for weight management may be prudent. Does the individual have concerns about needle-based treatments? In such instances, favoring orally administered medications could offer a more suitable solution. If there is an elevated risk of cardiac or renal complications, it would be wise to consider options from the SGLT2 inhibitor group or GLP1 agonist group. In essence, treatment needs to be tailored to the unique needs and circumstances of each individual, ensuring that treatment decisions align with their specific health challenges and objectives.

BARIATRIC SURGERY

Weight reduction plays a crucial role in the management of T2D because weight loss is associated with improved insulin resistance and glycemic status. Considering the limited efficacy of lifestyle and pharmacologic interventions in treating severe obesity and obesity-related T2D, along with the remarkable weight reduction and remission of T2D observed in youth who undergo bariatric surgery the ADA recommends considering bariatric surgery for adolescents with T2D who have a BMI >35 kg/m²(68). The two most common bariatric surgeries performed in adolescents with T2D are laparoscopic sleeve gastrectomy (LSG) and laparoscopic Roux-en-Y gastric bypass (LRYGB).

Impact of Bariatric Surgery on Glycemic Control

Studies have demonstrated improvement in HbA1c across all age groups. In one study, sixty-four pediatric patients diagnosed with morbid obesity and T2D underwent LSG surgery. The patients’ ages ranged from 5 to 14 years old. Their BMI decreased from 44.6±9.3 to 34.8 ± 9.6 kg/m².Their HbA1c decreased from 6.0±0.8% prior to LSG surgery to 5.4±0.4%, 12 months post-surgery, a change of 10.9% (p = 0.001) (69). There was no significant difference in post-operative HbA1c between the age groups.

Impact of Bariatric Surgery on T2D Remission

Bariatric surgery in adolescents with T2D has a better outcome compared to medical treatment with metformin alone or in combination with rosiglitazone or intensive lifestyle intervention with insulin therapy given for glycemic progression (70). In a multicenter, nonrandomized, retrospective study of 202 obese adolescents with T2D (before the approval of new drugs for adolescents with T2D), 109 adolescents underwent surgery, and 93 adolescents received nonsurgical treatment (71(. In the surgery group, the remission rate for diabetes was 76%, compared to 6.5% in the medical treatment group. The remission was sustained for the two years of follow-up. Of note, LRYGB had better effects on weight loss and glycemic control than LSG.

Impact of Bariatric Surgery on Kidney Outcomes

In a three-year longitudinal study, kidney outcomes in youth following bariatric surgery were assessed. Improvement in albuminuria and GFR was observed in those with pre-operative kidney impairment. Among participants with a preoperative eGFR < 90 ml/min/1/73m², eGFR improved from a mean of 76 ml/min/1.73m² to 102 ml/min/1.73m². Similarly, in those with an elevated albumin-to-creatinine ratio at baseline (ACR ≥30mg/g), the median ACR decreased from 74 mg/g to 17 mg/g. Estimated GFR and albuminuria remained stable in those without any evidence of pre-operative impairment. The effect of bariatric surgery on diabetic kidney disease was also investigated in adolescents with severe obesity and T2D five years after surgery compared to adolescents receiving medical management in the TODAY study (72). Elevated albuminuria was present in 21% of those receiving medical management at baseline, and it increased to 43% at 5 years. Conversely, albuminuria decreased from 27% prior to surgery to 5% at the 5-year follow-up. In adjusted analyses, the medical group had 27-fold higher odds of diabetic kidney disease at 5 years compared to Teen-LABS participants (72-73).

Impact of Bariatric Surgery on CVD Events

In another analysis, the risk for CVD events in two cohorts of adolescents with T2D and severe obesity undergoing medical or surgical treatment of T2D was assessed. Thirty adolescents who underwent bariatric surgery were matched to 63 who had received metformin alone or in combination with rosiglitazone or an intensive lifestyle intervention, with insulin therapy given for glycemic progression. The BMI of those who underwent bariatric surgery was 54.4±9.5 kg/m², and that of the medical group was BMI 40.5±4.9 kg/m². Although the baseline likelihood of CVD events was higher in the group that underwent bariatric surgery, one year after the surgery, the event risk was significantly lower and sustained at 5-year follow-up, whereas medical therapy was associated with an increase in risk among adolescents with T2D and severe obesity (74).

Adverse Effects of Bariatric Surgery

Bariatric surgery in adolescents can lead to various potential complications beyond the first postoperative month. The most commonly reported include gastroesophageal reflux, incisional hernia, treatment failure requiring operative revision, and nutritional deficits (50). Less frequent complications include stomach cancer, liver necrosis, gallbladder disorders, pancreatic disorders, acute kidney failure, neuromuscular complications, skin complications, and rarely mortality (51).

Summary

In summary, bariatric surgery currently outperforms medical management of T2D in adolescents and is the most efficacious treatment for youth-onset T2D, albeit with significant risks and economic implications. However, given the rapidly emerging medical therapies for weight reduction in adults and youth, many of which (e.g., dual-incretin analogs, triple-G agonists (75)) are now achieving degrees of weight loss approaching those seen following surgery, the individual roles of surgical and medical treatment for youth-onset T2D will need continued reassessment in the coming years.

COMPLICATIONS AND COMORBIDITIES AMONG ADOLESCENTS WITH T2D

Large cohort studies investigating the incidence of complications in children and teenagers with T2D unequivocally demonstrated a higher incidence of complications, both compared to the incidence of complications in T2D in adults and in T1D in children. In a multicenter observational study conducted from 2011 to 2020, the cumulative incidence of diabetic complications was assessed in 500 adolescents who had participated in the TODAY study (76). After only 13 years from diagnosis, higher complication rates were observed compared to those reported for pediatric patients with T1DM or for adults with T2D. The first striking finding from this report is the participants’ poor glycemic control despite participation in a well-resourced clinical trial. While glycemic control in adolescents with T1DM tends to improve with age, a gradual deterioration occurred in adolescents with T2D throughout the follow-up period. Approximately 45.0% had HbA1c of at least 10%, and an additional 20% were in the range of 8-10%. Their BMI remained consistently in the range of 35.0 to 37.5 kg/m2. It is important to note that poor glycemic control and obesity are associated with early complications. The complications associated with T2D in children affect various systems (Figure 5).

Figure 5. Comprehensive Overview of Type 2 Diabetes (T2D) Complications in Adolescents. A comprehensive visual representation of the diverse range of complications affecting major physiological systems that can arise in adolescents with T2D.

On average, thirteen years after the initial diagnosis of T2D, incidence rates for hypertension, dyslipidemia, diabetic kidney disease, and neuropathy were 67.5%, 51.6%, 54.8%, and 32.4%, respectively. Additionally, retinopathy was present in 51% of the participants. One-fifth of the cohort (21.3%) had two complications, and 7.1% had three. There were seventeen serious cardiovascular events, including myocardial infarctions, six cases of congestive heart failure, three cases of coronary artery disease, and four stroke events. Additionally, six deaths were reported. This rapid development of complications was associated with severe insulin resistance and poor socioeconomic circumstances.

Hypertension

Hypertension, defined as blood pressure ≥ 95^th percentile for age, sex, and height or systolic BP ≥ 130/80 mm Hg, is often present at the time of diagnosis in adolescents with T2D. In a study of 391 children from Hong Kong under 18 years of age, 22.5% had hypertension at diagnosis. In the TODAY study, 11.6% were hypertensive at the time of diagnosis (77) and the cumulative incidence of hypertension was 67.5% at a mean age of 26.4±2.8 years and 13.3±1.8 years from diagnosis (78). A systematic review and meta-analysis of 31 international studies on 4363 children with T2D aged 6.5 to 21 years at diagnosis found a pooled prevalence of hypertension of 25.33% (95% CI, 19.57%-31.53%) (79).Male participants had a higher hypertension risk than female participants (odds ratio 1.42 [95% CI, 1.10-1.83]).

Initial treatment involves lifestyle modification, aiming to not only enhance glycemic control but also incorporate a diet that is low in sodium and abundant in fruits, vegetables, whole grains, low-fat dairy products, and lean proteins. The Dietary Approaches to Stop Hypertension (DASH diet) is highly recommended for its effectiveness in reducing high blood pressure and supporting weight loss. In addition to lifestyle modification, the ADA recommends starting Angiotensin-Converting Enzyme inhibitors (ACEis) or Angiotensin Receptor Blockers (ARBs). Due to the potential teratogenic effects, the use of reliable contraception should be encouraged in individuals of childbearing age (68). There are no agents specifically approved for use in youth with T2D, however, a once-daily medication is recommended for better compliance (80,81).

Nephropathy

The primary microvascular diabetic complication in adolescents with T2D is diabetic kidney disease (DKD), which develops in 25–40% of T2D patients and is associated with rapid progression and poor prognosis. The severity and risk of rapid deterioration in young people with T2D are reflected in the fact that microalbuminuria is reported at diagnosis among adolescents with T2D. In a recent systematic review and meta-analysis (79) that included 14 studies of 2250 children and adolescents with T2D, the prevalence of albuminuria was 22.17% (95%CI, 17.34%-27.38%), and the pooled prevalence of macroalbuminuria among 730 children and adolescents was 3.85% (79). In the SEARCH study (82) after a duration of 8 years, the prevalence was 19.9% among adolescents with T2D compared with 5.8% in those with T1D.

The accelerated development of kidney damage is attributed to inadequate glycemic control and the presence of various risk factors, including obesity, dyslipidemia, insulin resistance, hypertension, elevated serum uric acid, female sex, and the presence of chronic inflammation (83).

While albuminuria is the earliest clinical sign of diabetic kidney disease (DKD), the natural history of DKD begins with hyperfiltration, characterized by an increase in glomerular filtration rate (GFR) >120 mL/min/1.73 m² as a consequence of obesity and impaired glucose tolerance (84). This increase predicts deterioration before other clinical signs appear. The second stage in the evolution of kidney dysfunction, still without clinical manifestations, is a mild reduction in GFR (60-89 mL/min/1.73 m²) (Figure 6). Structural changes of the kidney are typical at this stage, yet they are often reversible, making this a critical time for risk factor reduction (84).

Figure 6. Heat map depicting diabetes kidney disease (DKD) staging by GFR and albuminuria and suggested treatments. Adapted from Naaman SC, Bakris GLJDC. Diabetic Nephropathy: Update on Pillars of Therapy Slowing Progression. 2023;46:1574-86.

Treatment in these early stages involves achieving adequate glycemic control along with lifestyle modification, including increasing physical activity, improving sleep quality, and quitting smoking. Additionally, a healthy diet with lower salt and potassium intake, along with a low protein intake of 0.8 g/kg/day, is recommended (68). Pharmacological treatment includes control of blood pressure and managing dyslipidemia. Theoretically, medications that improve glycemic control would also provide benefits for preventing and/or treating DKD. GLP1 receptor agonists are reported to have a renal protective effect in adults with T2D. A recent meta-analysis of approximately 60,000 adults with T2DM treated with GLP-1 receptor agonist found a significant reduction in the composite kidney outcome (development of macroalbuminuria, doubling of serum creatinine, 40% or greater decline in eGFR, kidney replacement therapy, or death from kidney disease) when compared to placebo, as well as a trend towards a reduction in worsening kidney function (84). Similarly, the hemodynamic and natriuretic effects of SGLT2 inhibition are also postulated as protective mechanisms for DKD (85).However, studies in adolescents with T2D did not show an effect of systolic or diastolic blood pressure (Table 3). Of note, these studies involved relatively small groups of youth, and were short-term. Larger, long-term studies are needed, as well as additional therapeutic options for reducing the risk of DKD, such as mineralocorticoid receptor antagonists (MRAs), and endothelin receptor antagonists (ERAs).

Dyslipidemia

Screening for dyslipidemia should be conducted once optimal glycemic control has been achieved or within 3 months after T2D diagnosis. Initial cholesterol screening can be performed non-fasting. The recommended target values are triglycerides <150 mg/dL (1.7 mmol/L), HDL cholesterol >35 mg/dL (0.91 mmol/L), and LDL cholesterol <100 mg/dL (2.6 mmol/L). If these levels are outside the normal range, medical nutritional therapy (MNT) should be initiated, including restricting caloric intake from fat to 20–30% and daily cholesterol intake to <200 mg/day, avoiding trans fatty acids, limiting saturated fats to <7%, and controlling mono and poly unsaturated fats to 10-15%. If, after 6 months of dietary intervention, the LDL-C remains >130 mg/dL, statin therapy should be initiated, with a goal of achieving LDL-C <100 mg/dL. Currently, there are 7 approved statins for use in children and adolescents: Lovastatin, Simvastatin, Atorvastatin, and Fluvastatin [children ≥10 years old], Pitavastatin and Pravastatin [> 8 years], and Rosuvastatin [>6 years old]. It is important to note that due to potential teratogenic effects, individuals of childbearing age should receive reproductive counseling, and statins should be avoided in individuals of childbearing age who are not using reliable contraception. If triglyceride levels are greater than 400 mg/dL (4.7 mmol/L) in a fasting state or exceed 1,000 mg/dL (11.6 mmol/L) in a non-fasting state, it is imperative to optimize glycemic control and initiate fibrate therapy. This is essential for reducing the risk of pancreatitis.

In a study assessing whether clinicians are sufficiently aggressive in treating diabetes-related hyperlipidemia in youth, among 278 youth with T2D <10 years, 57% had LDL-C exceeding 100 mg/dL, 24% had LDL-C at or above 130 mg/dL, and 9% had LDL-C surpassing 160 mg/dL (86). Additionally, 29% had hypertriglyceridemia, and 44% had HDL-C levels below 40 mg/dL. However, only 5% of these youth were prescribed lipid-lowering medications.

Polycystic Ovarian Syndrome (PCOS)

There is a strong association between T2D in adolescent girls and polycystic ovary syndrome (PCOS). A systematic review and meta-analysis comprising 6 studies that involved 470 girls diagnosed with T2D, with an age range at diagnosis between 12.9 and 16.1 years, revealed that the prevalence of PCOS was estimated at 19.58% (87). PCOS is associated with a range of metabolic diseases, including hypertension and dyslipidemia. Furthermore, it is associated with a higher prevalence of cardiovascular risk factors, such as higher carotid intima thickness, β stiffness index, and reduced arterial compliance. Additionally, adolescent girls with PCOS have an increased likelihood of experiencing psychiatric comorbidities, such as anxiety and depression, which can negatively impact their health-related quality of life. A menstrual history should be taken on every girl with T2D at the diagnosis and at every follow-up visit. Metformin, in addition to lifestyle modification, is likely to improve menstrual cyclicity and hyperandrogenism in female individuals with T2D.

Pregnancy and Contraception

In the TODAY study, the maternal and offspring outcomes of young women with youth-onset T2D who experienced one or more pregnancies were evaluated (88). Over a span of up to 15 years, a total of 260 pregnancies were reported by 141 women. Their average age was 21.5±3.2 years, a mean BMI of 35.6±7.2 kg/m², and an average diabetes duration of 8.1±3.2 years (88). Complications during pregnancy were identified in 65% of the women. Elevated HbA₁c levels of ≥8% were observed in 31.9% of the pregnancies, and chronic hypertension complicated 35% of them. Pregnancy loss was observed in 25.3% and preterm birth occurred in 32.6% of pregnancies. Among the offspring, 7.8% of the babies were classified as small for gestational age, 26.8% as large for gestational age, and 17.9% fell into the macrosomic range. Other complications and congenital anomalies were noted in 10% of infants, including anencephaly, renal anomalies, and complications related to prematurity. The rate of known miscarriage for the entire study was 12.3%. The rate of known stillbirths in the cohort was 3%, which is more than triple the reported national rates.

The impact of T2D during pregnancy extends beyond the immediate prenatal and birth periods, exerting long-term effects on offsprings. Data pertaining to the diagnosis of diabetes in biological mothers were available for 621 participants in the TODAY study, of whom 301 had never been diagnosed with diabetes, 218 were diagnosed either before or during pregnancy, and 102 were diagnosed after pregnancy (89). For biological fathers, the data were available for 519 participants, with 352 having no history of diabetes and 167 having paternal diabetes. Notably, it was found that maternal diabetes, but not paternal diabetes, was associated with lower beta-cell function and deterioration of glycemic control over time. These effects remained significant even after accounting for variables such as age, sex, race/ethnicity, and household income (89).

Contraception is indicated in adolescent girls with T2D for several compelling reasons. Firstly, it is a recommendedtreatment for managing the symptoms of PCOS. Secondly, these young women are at increased risk of unintended pregnancy, which can lead to poor outcomes. Lastly, it is imperative for those treated with potentially teratogenic medications such as ACEis, ARBs, and statins. Unfortunately, despite the evident need, preconception counseling was reported only in 16.3% of women prior to their first pregnancy and only 14.9% used any method of contraception prior to the first pregnancy (88). This gap in reproductive health care highlights the importance of healthcare providers and support staff addressing this issue promptly and effectively.

Although in theory, adolescent girls with T2D without severe obesity, micro- or macrovascular disease, or other cardiovascular risk factors can consider a wide range of contraceptive methods, the practical reality often differs. A significant proportion of these individuals are afflicted by severe obesity, and a considerable number also exhibit elevated blood pressure. Consequently, when morbid obesity, severe hypertension, micro- or macrovascular disease, or multiple cardiovascular risk factors are present, it is advisable to prioritize nonhormonal or progestin-only contraceptive methods (Figure 7) (90).

Figure 7. Heat map of contraceptive treatment options for adolescent girls with T2D based on disease duration and complications. Adapted from Merino PM, Codner E. Contraception for Adolescents and Young Women with Type 2 Diabetes-Specific Considerations. Current diabetes reports 2022;22:77-84.

Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most important causes of visual loss worldwide. DR is often asymptomatic until the very late stages. Given the potential for a rapid rate of progression and the effectiveness of new therapies in slowing deterioration, it is imperative to routinely screen individuals with diabetes for the early detection of retinal changes. It is important to note that direct fundoscopy may be less sensitive than 7-field stereoscopic fundus photography for detecting retinopathy (91). Diabetic retinopathy (DR) is divided into two major forms: non-proliferative and proliferative, according to the presence of abnormal new blood vessels emanating from the retina. Non-proliferative DR consists of microvascular abnormalities (including microaneurysms, occluded vessels, and dilated or tortuous vessels) primarily in the macula and posterior retina, intraretinal hemorrhages, hard exudates; and the variable presence of nerve-fiber layer infarcts (cotton wool spots). Visual loss is mainly due to the development of macular edema. Proliferative DR is marked by the presence of neovascularization arising from the disc and/or retinal vessels, resulting in preretinal and vitreous hemorrhage, subsequent fibrosis, and traction retinal detachment resulting in visual loss.

In a meta-analysis that included 27 studies involving 5924 children and adolescents (91) with T2D duration between 6.5-21.0 years, 1.11% had DR less than 2.5 years after T2D diagnosis. The prevalence of DR increased over time, with rates of 9.04% at 2.5 to 5.0 years after T2D diagnosis, and 28.14% more than 5 years after T2D diagnosis. The global prevalence of diabetic retinopathy in pediatric T2D was found to be 6.99%. Optical coherence tomography (OCT)performed in adolescents with T2D in the TODAY study revealed that changes in retinal thickness correlated with HbA1c, fasting glucose, and blood pressure, illustrating the importance of intensive control of hyperglycemia, hypertension, and hyperlipidemia.(92)

Among the systemic strategies for the prevention and treatment of diabetic retinopathy, two randomized clinical trials, "The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study and the Action to Control Cardiovascular Risk in Diabetes (ACCORD)-Eye Study, documented a reduction in the risk of non-proliferative DR progressing in individuals using fenofibrate (93). The protective effects of fenofibrate have been attributed to its antioxidative, anti-inflammatory, anti-apoptotic, and anti-angiogenic properties. Other treatments include pan-retinal photocoagulation, intravitreal anti-vascular endothelial growth factor (VEGF) therapy, integrin antagonists, and anti-inflammatory agents. Corticosteroids remain important second-line therapies for patients with macular edema.

Diabetic Neuropathy

DIABETIC PERIPHERAL NEUROPATHY

Diabetic peripheral neuropathy (DPN) is a cause of significant disability and poor quality of life. Signs and symptoms include sensory loss, paresthesia, and pain. However, subclinical signs of DPN may precede the development of frank neuropathic symptoms, and systematic screening is required to identify DPN in its earliest stages. Studies from USA (78,94), Canada (95), and India (96) reported the prevalence of DPN in adolescents with T2D assessed by examination for foot abnormalities, distal vibration perception, and ankle reflexes (97). In the SEARCH study, the prevalence of peripheral neuropathy assessed after 8 years in 258 adolescents with T2D was 22%, compared with 7% in T1DM (98). Data from the TODAY Study Group revealed that at baseline, 1.0% of the participants had nerve disease, and the cumulative incidence at 15 years was 32.4% (78). The risk factors associated with peripheral neuropathy in youth are longer duration of diabetes, older age, male sex, and smoking.

Calcium channel a2δ ligands (gabapentin, pregabalin= Lyrica), serotonin- norepinephrine reuptake inhibitors, and tricyclic antidepressants are the most widely used medications for painful DN in adults. These medications are used to treat various presentations of neuropathic pain in children, but there have been no clinical studies devoted to painful DPN and no agents are licensed specifically for painful DN in childhood and youth (99). “Fortunately, painful DPN is relatively rare in children and youth.

AUTONOMIC NEUROPATHY

Diabetic autonomic neuropathy (DAN) is a common form of neuropathy in individuals with diabetes mellitus characterized by dysfunction due to impairment of peripheral autonomic nerves, with a wide spectrum of manifestations (100). DAN that involves autonomic fibers of the enteric nervous system may cause upper and lower gastrointestinal symptoms, including gastroesophageal reflux disease, gastroparesis, and constipation. Additional manifestations include bladder and sexual dysfunction. DAN also involves the cardiovascular system. Cardiovascular autonomic neuropathy (CAN), secondary to the pathology of the autonomic nerve fibers that innervate the heart and blood vessels, results in resting tachycardia, exercise intolerance, orthostatic hypotension, syncope, and silent myocardial infarction and ischemia(101). A reduction in heart rate variability is an early sign of CAN and was reported in 47% of youth with T2D after a mean disease duration of only 1.7 years (102). In the SEARCH study, the prevalence of CAN assessed after duration of 8 years in 252 adolescents with T2D was 17% compared with 12% in T1DM (103). CAN was associated with elevated triglycerides and increased urinary albumin excretion.

Cardiovascular Complications

T2D is a major risk factor for cardiovascular disease (CVD), including myocardial infarction and stroke. Markers for early CVD include increased arterial stiffness, carotid intima-media thickness, an alteration in the vascular endothelium, and cardiac autonomic neuropathy.
Arterial stiffness prevalence was significantly higher among youth with T2D than those with T1D (104). Arterial stiffness (measured by pulse wave velocity from carotid-femoral, femoral-foot, and carotid-radial), was measured in 388 youth with T2D at a mean age of 21 years and a diabetes duration of 7.7±1.5 years (105). Higher arterial stiffness was associated with an adverse change in left ventricular diastolic function. Similarly, cardiac parameters were assessed in 177 individuals with T2D, with a mean age of 24.6±4.2 years and a disease duration of 10.3±3.5 years (106). Of these, 72% were females, and their HbA1c levels averaged 8.9±1.9. Their parameters were compared to those of individuals with T1D. The prevalence of increased atrial stiffness was significantly higher in those with T2D, at 75.7%, compared to only 23.4% in individuals with T1D. In addition, the prevalence of cardiac autonomic neuropathy was significantly higher in those with T2D, at 16.9%, compared to 9.0% in individuals with T1D. Participants with T2D exhibited a higher left ventricular mass index, lower systolic function, and lower diastolic function compared to those with T1D.

Adolescents with T2DM have increased carotid intima-media thickness (cIMT) compared to obese and normal weight groups; the group difference was detected early at 14 years of age (107).

Vascular endothelial function was lower in adolescents with T2D compared to normal weight and compared to adolescents with T1D (108).These changes are of importance as the rate of all adjudicated heart, vascular, and cerebrovascular events was 3.73 per 1000 person-years (78). There were 17 serious cardiovascular events, myocardial infarction [4 events], congestive heart failure [6 events], coronary artery disease [3 events], and stroke [4 events].

Decreased Bone Mineral Density (BMD)

In a study involving 17 adolescents newly diagnosed with T2D and 59 age, sex, and BMI-matched controls, bone mineral density (BMD) was assessed at multiple sites while accounting for potential confounding factors such as age, sex, Tanner stage, and BMI (109). The results revealed that BMD Z-scores for the femoral neck and bone mineral apparent density Z-scores of the lumbar spine were notably lower in individuals with T2D compared to their healthy counterparts.In another cross-sectional study of BMD in 180 youths aged 10 to 23 years, the participants were categorized into three groups: those with T2D were compared to those with obesity (n=226) and those with a healthy weight (BMI <85th percentile; n=238) (110). An age-dependent pattern emerged, wherein the BMD Z-score in children was higher in the T2D group compared to the obese group. However, in adolescents and young adults, the BMD Z-scores were lower in the T2D group when compared to the obese group. These findings suggest that T2D may have a detrimental impact on bone density, particularly around the age when individuals reach peak bone mass. Considering the elevated fracture risk observed in adults with T2D, the decrease in bone density at a young age raises concerns about potential long-term morbidity and fracture risk in adulthood.

Mental Health Comorbidity

DEPRESSION

As part of routine diabetes care, a total of 197 adolescents and youth diagnosed with T2D, with a mean age of 16.9 years and 57% male, completed the Patient Health Questionnaire (PHQ) (111). 19.3% reported elevated depressive symptoms (PHQ score ≥10). Furthermore, in a sample of 53, 18.9% acknowledged thoughts of self-harm. Despite the prevalence of depressive symptoms, only 50.0% of those with depressive symptoms had a documented referral for mental health treatment in the electronic health record after the positive screening outcome. Older age, shorter diabetes duration, a higher HbA1c level, more blood glucose checks per day, and being prescribed oral medications were significantly associated with more depressive symptoms. Results from the TODAY study revealed that older teen girls had the highest rates of clinically significant depressive symptoms (112). Increased number of stressful life events were associated with elevated depressive symptoms. Depressive symptoms were correlated with low adherence to diabetes treatment, lower psychosocial functioning, and impaired health-related quality of life (QOL). In another study comparing PHQ between adolescents with T2D and T1D, the median PHQ-9 score in females with T2D was significantly greater than in females with T1D, but did not differ between males with T2D and T1D (113). The association between depression and T2D has been reviewed elsewhere (114).

DISORDERED EATING BEHAVIORS

In the TODAY study, out of 678 adolescents with T2D at a mean age of 14.0 years, 6% had clinical and 20% had subclinical levels of binge eating (115). Moreover, 50.3% of adolescents with T2D who are receiving insulin therapy had disordered eating behaviors (DEB) compared with 21.2% of those with T1DM (116). Disordered eating behavior is associated with poorer clinical outcomes and psychosocial well-being.

Mortality

In the SEARCH study, over a median follow-up of 5.3 years, the mortality rate among individuals with T1D was 70.6 deaths per 100,000 patient-years, compared with 185.6 deaths per 100,000 patients-years for those with T2D (117).^.When compared to the general populations of US states, it was observed that the mortality rate for individuals with T2D was significantly higher than expected while this was not the case for individuals with T1DM. Females had higher mortality rates than males. Among adolescents with T2D, the leading underlying cause of death was attributed to transport/motor vehicle accidents followed by accidental poisoning, and intentional self-harm. According to a life expectancy model, it was predicted that youth with T2DM lose approximately 15 years (118).

CONCLUSION

A summary of the key points is presented in Table 4.

Table 4. Key Points
• The prevalence of type 2 diabetes (T2D) varies across different countries and regions globally, but there is a consistent upward trend in its occurrence. Large-scale national studies in countries like Sweden, Germany, and India have reported similar increasing trends. • Contrary to common perception, not all adolescents with T2D are severely obese. Only 70-80% fall into this category. • Socioeconomic status plays a significant role in the prevalence of T2D, with lower socioeconomic status being associated with higher incidence in Western countries, while the opposite is observed in low-income countries. • Although adolescents are at higher risk of developing T2D, there are growing reports of T2D cases in children younger than 10 years old. • The gender distribution of T2D varies, with a higher proportion of cases in females in Western countries but a higher proportion in males in the Middle East and China. • Promisingly, there are new medications designed for weight loss, which hold the potential to reduce the high rates of complications associated with T2D in children. • There are low prescription rates for statins and contraception in the management of T2D in adolescents, highlighting potential gaps in medical care. • Many complications of T2D can potentially be reversed if detected and treated aggressively in their early stages.

Table 4. Key Points

• The prevalence of type 2 diabetes (T2D) varies across different countries and regions globally, but there is a consistent upward trend in its occurrence. Large-scale national studies in countries like Sweden, Germany, and India have reported similar increasing trends.

• Contrary to common perception, not all adolescents with T2D are severely obese. Only 70-80% fall into this category.

• Socioeconomic status plays a significant role in the prevalence of T2D, with lower socioeconomic status being associated with higher incidence in Western countries, while the opposite is observed in low-income countries.

• Although adolescents are at higher risk of developing T2D, there are growing reports of T2D cases in children younger than 10 years old.

• The gender distribution of T2D varies, with a higher proportion of cases in females in Western countries but a higher proportion in males in the Middle East and China.

• Promisingly, there are new medications designed for weight loss, which hold the potential to reduce the high rates of complications associated with T2D in children.

• There are low prescription rates for statins and contraception in the management of T2D in adolescents, highlighting potential gaps in medical care.

• Many complications of T2D can potentially be reversed if detected and treated aggressively in their early stages.

ACKNOWLEGEMENTS

The authors extend their heartfelt gratitude to the gifted illustrator, Mrs Noah Levek, for her exceptional eye-opening illustrations.

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Diabetes in People Living with HIV

ABSTRACT

People living with HIV (PLWH) are living longer and also have unique risk factors for developing several metabolic diseases, including diabetes. This has been observed in both high income and low to middle income countries. Risk factors for diabetes in PLWH consist of specific antiretroviral therapies (ART), including older generation protease inhibitors and nucleoside reverse transcriptase inhibitors, lipodystrophy, and hepatitis C co-infection. In addition, obese and overweight states are common in PLWH, and an increased risk of incident diabetes has been noted with weight gain after ART initiation in PLWH, compared to individuals without HIV. Inflammation associated with HIV has also been linked to incident diabetes. This chapter reviews points to consider in the diagnosis and monitoring of diabetes in PLWH and discusses interactions that may occur between specific ART agents and glucose-lowering medications. Moreover, PLWH have risk factors for complications involving organ systems that are also affected by microvascular disease in diabetes. Because PLWH have a greater risk for cardiovascular disease (CVD) than individuals without HIV, modifiable risk factors of CVD should be addressed in the care of PLWH, considering that dyslipidemia, hypertension, and cigarette smoking are all highly prevalent in PLWH.

BACKGROUND

HIV infection is prevalent in 38.4 million people worldwide as of 2021 (1) and 1.2 million people in the United States as of 2019. The incidence of HIV infection has decreased from 2015 to 2019 in the United States but has remained stable in some demographic groups, including African-Americans and Latinos (2). Untreated, HIV infection can lead to opportunistic infections including cytomegalovirus disease and pneumocystosis (3) associated with AIDS, which is defined as a CD4+ cell count < 200 cells/mL. With advances in antiretroviral therapy (ART), however, HIV infection has been transformed from a disease strongly linked to AIDS and opportunistic infections into a chronic disease that is associated with several cardiometabolic consequences, including diabetes (4), heart disease (5), and other non-AIDS related illnesses such as osteoporosis (6). In part, this phenomenon is secondary to the fact that people living with HIV (PLWH) are living longer and are more susceptible to diseases of aging.

An important aspect to consider in the understanding of HIV associated cardiometabolic diseases is their growing global presence. The same factors that are associated with cardiometabolic disease prevalence in PLWH in high-income countries are also present in low-income countries, in addition to unique factors including industrialization, with resultant decreased physical activity and increased energy consumption (7) (8). As such, the care of PLWH needs to focus not only on ART but also on the management of chronic co-morbidities. This chapter will focus primarily on diabetes in PLWH.

EPIDEMIOLOGY AND RISK FACTORS FOR DIABETES IN PLWH

Epidemiology

PLWH have a unique set of risk factors that may increase their likelihood of developing diabetes. Illustrating the greater burden of diabetes in PLWH on ART, a study from 2005 on the Multicenter AIDS Cohort Study (MACS) of gay and bisexual men with (MWH) and without HIV (MWoH) found that the incidence of diabetes was found to be more than fourfold higher in MWH and that the prevalence of diabetes was 14% in MWH on ART and 7% in MWH not on ART, compared to 5% in MWoH. These differences were significant even after adjusting for age and body mass index (BMI). Of note, the majority of MWH on ART in the study were on first generation protease inhibitor (PI) therapy (discussed further in the section Protease Inhibitors) (9). Other studies have described incidence rates of diabetes in PLWH of 4.4 cases per 1000 person-years of follow-up in the Swiss HIV Cohort Study (10) and 5.72 cases per 1000 person-years of follow-up in the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) Study of participants from Europe, the US, Australia, and Argentina (11).

The effect of HIV disease on developing diabetes is also growing in low- and middle-income countries (LMIC), where the majority of the people with diabetes live (12). Prevalence of estimates of diabetes in PLWH in LMIC range from 6.8% in Chile (13) to 26% in Cameroon (14). PLWH and diabetes in LMIC are younger than those in high income countries (15). The wide range of prevalence estimates of diabetes in PLWH in LMIC may be in part because of differences in the definitions used to identifying diabetes. In addition to factors common to PLWH globally, urbanization may be a contributing factor to the development of diabetes in PLWH in LMIC (16).

Antiretroviral Therapy (ART)

PROTEASE INHIBITORS (PI)

One risk factor for developing diabetes in PLWH is PI use. In 1995, saquinavir became the first FDA approved PI (17). Incidence rates of hyperglycemia as high as fivefold have been reported in the setting of PI use (18). In addition, PIs, including ritonavir, have been associated with hypertriglyceridemia (19,20) and lipodystrophy (21). Mechanisms that may account for these effects are multifold. In one study, insulin sensitivity, as measured by glucose infusion rate during hyperglycemic clamp, decreased significantly after 12 weeks of PI therapy compared to baseline. A defect in beta cell function, in particular, a decrease in the disposition index, was also observed after PI therapy (22). An in vitro study demonstrated that indinavir may lower the function of the glucose transporter GLUT4 (23). Additional mechanisms may include changes in the hormone adiponectin, which is associated with improved insulin sensitivity, although in vivo and in vitro effects of PIs on adiponectin have differed (24,25).

The prescribing patterns of PI use have evolved. One study found that in the Veterans Affairs system, the prevalence of PI based regimen use decreased from 1997 to 2004 (26). In addition, the effects of all PIs, namely older generation versus newer generation PIs, including atazanavir and darunavir, on glucose homeostasis are not equal. In an in vitro study, atazanavir, which received FDA approval in 2003 (27), did not inhibit either GLUT1 or GLUT4 glucose transporters (28). In a clinical study of HIV- participants, atazanavir in combination with ritonavir had a significantly lower effect on insulin sensitivity compared to lopinavir/ritonavir, as measured by glucose disposal rate during hyperinsulinemic euglycemic clamp. (29). Moreover, the newer generation PIs have not been associated with an increased incidence of diabetes, with one study showing a lower risk of diabetes in individuals on these medications (30).

NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NRTIs)

In addition to PIs, certain nucleoside reverse transcriptase inhibitors (NRTIs) have been associated with diabetes. In the D:A:D study, incident diabetes was associated with exposure to the NRTIs stavudine and zidovudine, both thymidine analogs, and didanosine, after adjusting for age, sex, race, and BMI (11). NRTIs have also been implicated in mitochondrial dysfunction, which may be another mechanism by which NRTIs are associated with diabetes. One study found that one month of treatment with stavudine was associated with significant decreases in mitochondrial DNA from muscle biopsies and insulin sensitivity, as measured by the glucose infusion rate during a hyperinsulinemic euglycemic clamp (31). Because of serious adverse effects linked to the use of these NRTIs, stavudine, zidovudine, and didanosine are not advised for use in the United States (32).

Similar to the newer generation of PIs, the use of newer NRTIs, including emtricitabine, abacavir, and tenofovir, have been associated with a lower risk of diabetes (30). Supporting this finding, tenofovir disoproxil fumarate (TDF), in a study of participants without HIV or diabetes, did not significantly affect insulin sensitivity, as measured by hyperinsulinemic euglycemic clamp (33).

Tenofovir alafenamide (TAF) is a newer prodrug of tenofovir and is associated with less renal and bone toxicity than TDF (33a). However, TAF has been associated with weight gain in PLWH, compared with TDF (33b). Weight gain leading to an overweight state or obesity is a risk factor for the development of type 2 diabetes. In a retrospective study of the ADVANCE trial, which included ART naïve PLWH randomized to 3 treatment arms, 2 of which included TDF and 1 of which included TAF, an increase in the 10-year predicted risk of type 2 diabetes was observed for participants in the TAF arm, compared to participants in one of the TDF arms, although the risk calculator that was used had not been validated for the population in the study (33c). On the other hand, in a cohort study of more than 4,000 PWH on a TDF-based ART regimen for at least 6 months, of whom about 80% switched to a TAF-based ART regimen and about 20% continued on a TDF-based ART regimen, TAF was associated with a significant weight gain, compared to TDF, after 18 months, but it was not associated with incident diabetes.

INTEGRASE STRAND TRANSFER INHIBITORS (INSTIs)

Integrase strand transfer inhibitor (INSTI) based regimens are among the recommended initial ART regimens for PLWH (32) (34). Reasons for this include the effectiveness of and fewer adverse effects associated with INSTIs in studies of treatment-naïve patients (34-37). However, studies have discovered some metabolic effects of INSTIs. These include weight gain on average of 2.9 kilograms over 18 months, as observed in a retrospective study of patients who switched to an INSTI based regimen from previously being on a non-NRTI (NNRTI)-based regimen. This was in contrast to an average 0.7 kg weight gain in those participants who switched to a PI-based regimen and 0.9 kg weight gain in those who continued on a NNRTI-based regimen (38). In addition to weight gain, case reports of new onset diabetes have been reported in conjunction with INSTI use (39,40). One clinical trial compared the INSTI raltegravir to 2 boosted PI regimens and found that the increases in homeostasis model assessment-insulin resistance (HOMA-IR) in all 3 arms were not significantly different from one another, were noted by 4 weeks, and appeared to be independent of changes in visceral adipose tissue (41). However, other studies have not demonstrated an association between diabetes and INSTI use (41a) or have found that the association between INSTI use and incident diabetes varies by individual INSTI (41b).

The exact mechanisms by which INSTIs may be related to weight gain and diabetes are not known. One proposed mechanism by which INSTIs may cause hyperglycemia is their effect on magnesium, which is a necessary cation for insulin action (40). In mice, INSTI use leads to an increase in fat mass and decreases brown and beige adipocyte differentiation, leading to lower energy expenditure and weight gain (41c).

Lipodystrophy

Lipodystrophy associated with ART is characterized by either lipoatrophy in the face, arms, and legs, or lipoaccumulation that can lead to gynecomastia, dorsocervical fat tissue, and increased intra-abdominal fat, (42,43), or mixed lipodystrophy, in which lipoatrophy and lipoaccumulation occur together. As noted above, lipodystrophy has been associated with PI use (21) and with older generation NRTI use, including stavudine (44), but less commonly with the newer generation NRTI tenofovir (45). In a cross-sectional study, HIV+ men with lipodystrophy had a greater insulin resistance than men without lipodystrophy (21). Lipodystrophy has also been associated with incident diabetes (46,47).

In one study that measured body fat changes longitudinally using dual-energy x-ray absorptiometry (DXA) in PLWH on ART (specifically zidovudine and lamivudine or didanosine and stavudine in combination with nelfinavir, efavirenz, or both), 32% of participants had discordant changes in trunk and limb fat (48).

Lipodystrophy may also be persistent in patients with exposure to thymidine analogs. Although improvements in limb fat mass were reported in patients who switched from taking the thymidine analog zidovudine to the NRTI abacavir, no significant improvement was noted in self-assessment of dorsocervical fat (49).

Hepatitis C Co-Infection

25% of PLWH in the U.S. are co-infected with hepatitis C (50). Hepatitis C infection is associated with a higher risk of developing diabetes. Treatments for hepatitis C, including direct-acting antiviral treatment and pegylated interferon/ribivirin, have been found to lower the incidence of diabetes, with a larger effect in those patients with advanced fibrosis/cirrhosis. In addition among individuals who received treatment for hepatitis C, those with a sustained virologic response were less likely to develop diabetes than those without a sustained virologic response (51).

Weight Gain, Overweight, and Obesity

Weight gain that is observed after the initiation of ART in those patients with wasting associated with untreated HIV has been characterized as a “return to health” phenomenon. However, because of an emphasis on early ART initiation, the wasting that was previously seen with advanced HIV infection is less common in countries with access to ART (52). In one 2012 study conducted in Alabama, more than 40% of patients were overweight or obese at the time of starting ART. After 2 years on ART, the percentages of underweight and normal weight participants decreased significantly, and significant increases in the percentages of overweight and obese participants were observed. Having a lower baseline CD4 count and the use of a PI as a third drug were risk factors associated with a greater increase in BMI (53).

The consequences of weight gain in PLWH may be different from those in the general population. In a study of U.S. Veterans, the risk of incident diabetes with 10 or more pounds of weight gain during the first year after ART initiation in HIV+ Veterans was significantly greater than in HIV- Veterans (54).

Among cohorts of PLWH from different countries, prevalence estimates of an overweight state or obesity range from 25% to 68% (55,56). In one study of PLWH, risk factors for being overweight or obese in PLWH included increasing age and either no evidence of hepatitis C infection or evidence of cleared hepatitis C infection (57).

Inflammation

In addition to the risk factors for diabetes in PLWH outlined above, inflammation may play a role in the development of diabetes in PLWH. Systemic inflammation results from several factors: viral replication, immune activation, which leads to T cell depletion, as well as T cell loss specifically in the gastrointestinal tract, with associated translocation of microbial factors including lipopolysaccharide (58). Co-infection with viruses, including hepatitis C virus (as discussed above), hepatitis B virus, Epstein-Barr virus, and cytomegalovirus, perpetuate systemic inflammation (59).

Although ART reduces some inflammatory biomarkers in PLWH (60), there is evidence of residual inflammation, with levels of other inflammatory biomarkers in PLWH on ART that do not decrease to levels seen in HIV- individuals (61). Persistent inflammation measured months after ART initiation has been associated with incident diabetes in PLWH (62).

DIAGNOSIS OF AND MONOTORING DIABETES IN PLWH

The American Diabetes Association (ADA) recommends the use of a fasting plasma glucose, hemoglobin A1c, or a 2-hour plasma glucose after a 75-gram oral glucose tolerance test to establish the diagnosis of diabetes in the general population. However, there is a caveat that for certain populations of patients, including PLWH, fasting plasma glucose is preferable to hemoglobin A1c (63). Hemoglobin A1c has been found to underestimate glycemia in PLWH (64,65). One reason for this is the use of medications that cause hemolysis, including dapsone and trimethoprim-sulfamethoxazole, which are used for prophylaxis of Pneumocystis jiroveci, an opportunistic infection (65). However, another reason for the discrepancy between glucose levels and hemoglobin A1c is NRTI use. NRTIs, especially the thymidine analogs, are associated with an increased risk of macrocytosis (66), which can lower hemoglobin A1c. Finally, lower CD4 cell count (< 500 cells/mm³) was associated with a significant discordance between expected and measured hemoglobin A1c in MWoH in the MACS (67). As such, self-monitoring of blood glucose in PLWH may be preferable to using hemoglobin A1c for monitoring glycemic control (64).

The most recent Infectious Diseases Society of America guidelines on the primary care of PLWH recommends obtaining either a fasting plasma glucose and/or hemoglobin A1c at baseline and at 1 to 3 months after starting ART. In addition, hemoglobin A1c is preferred to fasting glucose for diagnosing diabetes because of the relative ease of obtaining a hemoglobin A1c over a fasting glucose. However, a hemoglobin A1c level ≥ 5.8% can be considered to diagnose diabetes in PLWH, instead of the ADA recommendation of ≥ 6.5%, as this improves the sensitivity of the test (68,69). Moreover, a hemoglobin A1c is recommended to be obtained every 6 months in PLWH and diabetes, with a goal hemoglobin A1c of < 7% (68).

TREATMENT OF DIABETES IN PLWH

Special considerations should be taken into account in the treatment of diabetes in PLWH. PLWH may not respond to treatment for diabetes in the same manner as HIV- individuals. Part of this observation may be because treatment responses were measured using hemoglobin A1c, which may be an inaccurate indicator of glycemia in PLWH (70), as noted above.

Interactions between several diabetes medications and ART are known. In addition, some diabetes medications may present advantages or disadvantages in PLWH. These conditions are described in further detail below and are organized by diabetes medication.

Lifestyle Interventions

Both the 2023 ADA Standards of Care on Pharmacologic Approaches to Glycemic Treatment and the 2023 updated American Association of Clinical Endocrinology algorithm of the management of type 2 diabetes highlight lifestyle interventions as part of the recommended treatment of type 2 diabetes (70a, 70b). Lifestyle interventions include not only a healthy diet and exercise but also smoking cessation and moderating alcohol intake (70b).

Physical activity has benefits for PLWH, including decreasing waist circumference (70c) and improvements in glucose and high-density lipoprotein cholesterol levels (70d). However, low physical activity has been reported in some studies of PLWH (70e, 70f).

Metformin

The 2023 ADA Standards of Care on Pharmacologic Approaches to Glycemic Treatment includes metformin as one first-line treatment to consider in patients in the general population with type 2 diabetes (70a) (71).

In PLWH with lipodystrophy and insulin resistance but without diabetes, 3 months of metformin 1000 mg twice daily was found to significantly decrease insulin area under the curve after an oral glucose tolerance test. Although no increased incidence in lactic acidosis was noted in the participants who received metformin, the study was not powered for this outcome, and liver and kidney dysfunction were exclusion criteria (72).

The commonly used integrase transfer strand inhibitor, dolutegravir, increases plasma levels of metformin, and thus adjusting the dose of metformin upon dolutegravir initiation has been recommended (73,74). However, one retrospective study found that there was no significant difference in glycemic control before and after starting dolutegravir in PLWH and diabetes on metformin (75).

In summary, there are no guidelines to suggest that metformin should not be a possible first-line treatment of type 2 diabetes in PLWH, after consideration of liver and kidney function and dolutegravir treatment.

Sulfonylureas

Sulfonylureas are a substrate of the CYP2C9 enzyme. The PIs ritonavir and nelfinavir are CYP2C9 inducers and can decrease sulfonylurea levels (76,77). In comparison with initial use of metformin in PLWH, no significant difference in glycemia after one year of therapy was noted with initial use of a sulfonylurea (78).

The 2023 ADA guidelines note that sulfonylureas are a high efficacy drug class for glucose management and are less cost-prohibitive compared to many other drug classes (70a) (71). However, adverse effects of sulfonylureas include hypoglycemia and weight gain. In PLWH, consideration should be made if a patient is on ritonavir or nelfinavir and the possible loss of efficacy of sulfonylurea treatment.

Thiazolidinediones

The effect of thiazolidinediones on glycemic control in PLWH with diabetes specifically has not been studied, although some trials found that rosiglitazone lowered serum insulin levels in PLWH without diabetes (79) and improved insulin sensitivity in PLWH with hyperinsulinemia (80). Several studies have focused on the effect of thiazolidinediones (TZDs) on body fat in PLWH with lipodystrophy, with equivocal findings. One 24-week study on the effect of rosiglitazone on PLWH, all of whom were on a PI, found no significant difference in arm fat between the treatment and placebo groups (81). However, the study did not have enough power to detect a difference in this outcome (82). On the other hand, other studies demonstrated that rosiglitazone increased visceral and subcutaneous abdominal fat in MWH with lipodystrophy over 6 months (83) and subcutaneous leg fat in PLWH with lipodystrophy over 3 months (80).

Adverse side effects of TZDs should be considered and discussed prior to initiation. These include fluid retention, osteoporosis, edema, and potential liver injury (84).

Dipeptidyl Peptidase 4 Inhibitors

Some clinical studies have demonstrated that dipeptidyl peptidase 4 (DPP4) inhibitors exert an anti-inflammatory effect in patients with type 2 diabetes (85,86). This idea was further examined in PLWH in a pilot study of 20 PLWH on ART randomized to either the DPP4 inhibitor sitagliptin or placebo for 24 weeks. A significant decrease was noted in the chemokine SDF-1α in the treatment group. In addition, an improvement in glucose tolerance was noted at week 8 in the treatment group, but the difference in glucose tolerance between the two groups was no longer significant at the end of the study (87). In another study of PLWH with impaired glucose tolerance, sitagliptin resulted in a significant improvement in glucose tolerance and decreases in the inflammatory markers hsCRP and CXCL10 from baseline after 8 weeks of treatment, compared to placebo (88). Similarly, in a larger study of 84 PLWH on ART with viral suppression and without diabetes who were randomized to 16 weeks of sitagliptin versus placebo, a significant decrease from baseline was seen at week 15 in CXCL10 in participants in the treatment group (89).

Glucagon-Like Peptide-1 Receptor Agonists

Several glucagon-like peptide-1 (GLP-1) receptor agonists have been shown to improve weight and cardiovascular outcomes, albeit in patient populations not specific to PLWH (90,91). Liraglutide, dulaglutide, and semaglutide have been shown to lower the risk of a composite outcome of nonfatal stroke, nonfatal myocardial infarction, and cardiovascular death in patients with type 2 diabetes (90,91). Liraglutide, semaglutide, and dulaglutide have indications from the Food and Drug Administration for lowering cardiovascular event risk in patients with cardiovascular disease and type 2 diabetes, and dulaglutide is also approved for lowering cardiovascular event risk in patients with multiple cardiovascular risk factors and type 2 diabetes. The 2023 ADA guidelines recommend the use of a GLP-1 receptor agonist as a first-line treatment in patients with cardiovascular disease and type 2 diabetes (70a).

One case report described a single PLWH who was able to discontinue insulin treatment (insulin glargine 60 units daily) and who experienced improvements in weight and hemoglobin A1c after starting liraglutide therapy (92). Another case report described improvements in hemoglobin A1c and fasting glucose on dulaglutide in a PLWH. Relatively scant literature exists on the effects of GLP-1 receptors agonists in PLWH and diabetes (93,94).

Sodium-Glucose Co-Transporter-2 Inhibitors

Similar to the GLP-1 receptor agonists, sodium-glucose co-transporter-2 (SGLT2) inhibitors have demonstrated reductions in adverse cardiovascular outcomes, including cardiovascular death and hospitalization for heart failure in general population studies (95,96). In addition, both dapagliflozin and canagliflozin have an FDA indication to reduce the risk of end-stage renal disease in patients with type 2 diabetes and diabetic nephropathy with albuminuria (96a). As such, the 2023 ADA guidelines on pharmacologic treatment of diabetes recommend that in those patients with type 2 diabetes and chronic kidney disease, a SGLT2 inhibitor that has been shown to reduce risk of renal disease progression should be used (70a).

SGLT2 inhibitors are also approved for reduction of adverse cardiovascular events and heart failure hospitalization in people with diabetes and have cardiovascular benefits for patients with either systolic or diastolic heart failure (96a). Diastolic dysfunction is more common in PLWH compared with people without HIV. As such, the 2023 ADA guidelines recommend the use of an SGLT2 inhibitor with demonstrated benefit in patients with type 2 diabetes and heart failure (70a).

One small trial studied canagliflozin for 24 weeks in 8 obese PLWH with type 2 diabetes and hemoglobin A1c > 7% and observed improvements in weight and hemoglobin A1c at the end of the study compared to baseline (97).

Adverse side effects of SGLT2 inhibitors include genital mycotic and urinary tract infections. In addition, 55 post-marketing cases of Fournier’s gangrene have been reported between March 2013 to January 2019 (98). A risk factor for Fournier’s gangrene is HIV infection (99-101), especially a CD4 cell count < 200 cells/μL (102).

Insulin

The 2023 ADA guidelines recommend considering insulin initiation in the patient 1) with a hemoglobin A1c > 11%, 2) with signs and symptoms of catabolism, 3) who has a presentation concerning for type 1 diabetes, and/or 4) whose hemoglobin A1c is not at goal despite taking 2 to 3 medications for diabetes, in addition to a GLP-1 receptor agonist.

As with all patients with diabetes, side effects of insulin therapy to consider in a discussion with a patient with HIV disease and diabetes include the risks of hypoglycemia and weight gain.

Sequence of Initiating Diabetes Medications in PLWH

There are no specific guidelines for the treatment of diabetes in PLWH. The 2023 ADA guidelines recommend the treatment of type 2 diabetes be tailored to the individual patient and consider factors including cost of medications, weight loss goals, and comorbidities including chronic kidney disease, atherosclerotic cardiovascular disease, and heart failure. Healthy lifestyle interventions should be incorporated alongside the pharmacologic treatment of type 2 diabetes.

For those patients with type 2 diabetes and ASCVD or who are at high risk for ASCVD, treatment with either a GLP-1 receptor agonist or SGLT2 inhibitor is recommended. If A1c is not at goal, then adding an additional agent, 1) such as a SGLT2 inhibitor, if the patient is already on a GLP-1 receptor agonist, or a GLP-1 receptor agonist, if the patient is already on a SGLT2 inhibitor, or 2) a thiazolidinedione, is recommended (70a). For patients with type 2 diabetes and heart failure, a SGLT2 inhibitor is recommended, as noted above. For patients with type 2 diabetes and chronic kidney disease, a SGLT2 inhibitor, or if contraindicated, a GLP-1 receptor agonist is recommended. Finally, for those patients with type 2 diabetes for whom weight management is a priority, a medication with weight loss or weight neutral effects can be considered. With regards to weight loss, semaglutide and tirzepatide have the most effect, whereas metformin and DPP4 inhibitors are weight neutral. Dulaglutide and liraglutide have a high weight loss efficacy, and SGLT2 inhibitors have intermediate weight loss efficacy (70a).

In PLWH, an additional factor to consider is the possible interaction of a diabetes medication with specific ART.

MICROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

Peripheral Neuropathy

HIV is associated with multiple peripheral neuropathies (PNs), including a distal, symmetric polyneuropathy (DSPN) (103). The prevalence of HIV-associated PN among PLWH varies < 10% to upwards of 50%, with the wide variability in prevalence estimates secondary in part to differences in methods used to assess PN (104). Risk factors for HIV-associated DSPN include use of the NRTIs zalcitabine, didanosine, and stavudine (105). In addition, HIV-associated DSPN has been observed in PLWH within 1 year of HIV transmission and is significantly associated with evidence of immune activation present in the central nervous system (106).

HIV-associated PN, in addition to the risk of developing PN secondary to diabetes, may place PLWH and diabetes at increased risk of sequelae including falls, particularly in those PLWH with a detectable viral load (107), foot ulceration, and amputations.

In summary, in addition to considering the risk of diabetic peripheral neuropathy, the risk of HIV-associated PNs should be kept in mind in the assessment and treatment of PLWH with diabetes.

Nephropathy

The prevalence estimates of chronic kidney disease (CKD) in PLWH vary depending on geographic region, with 7.9% of PLWH affected in Africa (with the highest prevalence among African regions in West Africa at 22%), compared to 3.7% in Europe. In addition, prevalence estimates of CKD in PLWH are significantly greater among those individuals with co-morbid diabetes (108). In the MACS, two independent risk factors associated with greater odds of proteinuria were HIV+ serostatus with ART use, compared to HIV- serostatus, and a history of diabetes (109). Moreover, glomerular hyperfiltration, or a supranormal estimated glomerular filtration rate, is more prevalent among HIV+ men without CKD than HIV- men without CKD (110). Glomerular hyperfiltration has been reported to be an initial state of dysfunction seen in patients with diabetic kidney disease and proteinuria (111).

Other risk factors for CKD in PLWH include the following: recurrent acute kidney injury, African-American race, in part because of risk variants of the APOL1 gene, and persistent inflammation, even in the setting of ART (112-114). In addition, an HIV-associated nephropathy (HIVAN), which is characterized by several insults to the kidney, including focal and segmental glomerulosclerosis, exists. Certain ART drugs within the NRTI, NNRTI, and PI classes are also associated with renal injury (114). Among these are the NRTI tenofovir, which is associated with adverse kidney disease outcomes independent of diabetes (115), and the PI indinavir (116).

Retinopathy

Ocular opportunistic infections in PLWH include CMV retinitis and ischemic HIV retinopathy (117). In addition, patients with a history of CMV retinitis should be monitored periodically for retinitis recurrence by an ophthalmologist (118). However, these ocular opportunistic infections are less common in the setting of ART. However, retinal disease has been noted in PLWH on ART as part of a larger syndrome of the HIV-associated neuroretinal disorder (117), which has an incidence of more than 50% at 20 years after a diagnosis of AIDS (119). One study found that among HIV+ men with a median duration of ART use of 12 years and suppressed viremia did have a significant difference in total peripheral retinal thickness from HIV- men, although the long-term clinical relevance of this is unknown (117).

There is limited literature on the effect of co-morbid diabetes on HIV-associated retinal disease.

MODIFIABLE FACTORS OF MACROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

PLWH are at increased risk of developing atherosclerotic cardiovascular disease (ASCVD) compared to individuals without HIV, despite controlling for traditional cardiovascular (CV) risk factors such as diabetes (5,120). In addition, PLWH have a greater burden of traditional CV risk factors (120). Other non-traditional risk factors include some ART such as older generation PIs (121) and inflammation (122). As such, calculators developed for the general population to calculate ASCVD risk may not accurately capture risk in PLWH (123).

Aspirin

Recent American College of Cardiology/American Heart Association (ACC/AHA) recommend the use of aspirin for primary prevention of ASCVD in general population patients age 40 to 70 years with high ASCVD risk (124), and similarly the American Diabetes Association (ADA) guidelines also recommend aspirin use in select patients with diabetes < 70 years of age with high ASCVD risk and low bleeding risk (125). However, evidence has shown that aspirin use is lower in PLWH with CV risk factors than in HIV- individuals (126), and PLWH are less likely to be prescribed aspirin for primary prevention than HIV- individuals (127).

Blood Pressure

The prevalence of hypertension globally in PLWH is substantial. PLWH have several risk factors for developing hypertension, including a greater prevalence of smoking (as noted below) (128,129) and ART use (130). The relationship of ART to hypertension is thought to be in part from its association to weight gain. Moreover, in the MACS, ART use was associated with greater systolic hypertension but not diastolic hypertension. A mechanism for this could be a change in arterial compliance as a consequence of ART use (131).

The 2023 ADA guidelines recommend a goal blood pressure of < 130/80 (132).

Cholesterol

Dyslipidemia is seen in both untreated PLWH and PLWH on ART (133,134). The decision to initiate a statin in a patient living with HIV for primary prevention should take into account the patient’s HIV serostatus, especially in those patients with a 10-year ASCVD risk of 5 to < 20% (124).

The primary prevention of atherosclerotic cardiovascular disease is a clinically important topic. The Evaluating the Use of Pitavastatin to Reduce Cardiovascular Disease in HIV-Infected Adults (REPRIEVE) study is a multicenter, international, randomized clinical trial on PWH on ART, in which participants with a low to moderate risk of ASCVD were randomized to either pitavastatin daily or placebo (134a). In March 2023, the Data Safety and Monitoring Board recommended premature closure of the study based on the observed efficacy of the study treatment to reduce the primary endpoint of major adverse cardiovascular events (MACE) by 35% relative to the placebo. The incidence of a major adverse cardiovascular event was 4.81 per 1000 person-years in the pitavastatin group and 7.32 per 1000 person-years in the placebo group (hazard ratio, 0.65; 95% confidence interval, 0.48 to 0.90; P = 0.002). Of note, similar to other studies the risk of diabetes was increased in the pitavastatin group (diabetes mellitus occurred in 5.3% in pitavastatin group and 4.0% in the control group. These results suggest that many PLWH should be on statin therapy.

Cigarette Smoking

Cigarette smoking is more prevalent in PLWH than in individuals without HIV (129), and life expectancy is estimated to be lower in HIV+ men and women who are current smokers at the time of HIV care initiation than in HIV+ men and women who are former or never smokers, with a 2.9 year gain in life expectancy at 10 years after HIV care initiation (135). The ADA guidelines recommend that people with diabetes not smoke cigarettes and that smoking cessation treatment be offered for those patients with diabetes who do smoke (136).

SUMMARY

In summary, PLWH have unique risk factors that increase their risk of diabetes. Factors to take into consideration in the treatment of PLWH include the following: type of ART, evidence of lipodystrophy, co-infection with other viruses, and overweight or obese state. A caveat is that HbA1c may be inaccurate in diagnosing and monitoring diabetes in PLWH. PLWH have additional risk factors for developing microvascular complications of diabetes. Some glycemic agents may interact with ART, and other glycemic agents may have unwanted effects, including weight gain, that should be addressed in a patient-provider discussion. Finally, additional modifiable cardiovascular risk factors, including hypertension and smoking, should be addressed in the comprehensive treatment of diabetes in PLWH.

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