Special Considerations Relevant to Pediatric Obesity

ABSTRACT

In most humans, body fatness is a quantitative trait reflecting the interactions of environment, genotype, and development. The metabolic predisposition to obesity and its co-morbidities in adulthood begins in the intrauterine environment, extends into early childhood, and is further impacted by puberty. An understanding of the pathogenesis of obesity in children, and its implications for the risk of obesity in adulthood, has the potential to inform healthcare providers about early identification and use of precision medicine approaches towards both prevention and treatment. This chapter begins with a review of the epidemiology and definition of pediatric obesity followed by a discussion of risk factors for adult obesity from genetics to the prenatal environment (epigenetics) through childhood. The next section emphasizes that while some adiposity-related problems are unique to the pediatric population, multi-system co-morbidities of adult obesity are increasingly prevalent in children. The chapter concludes with a discussion of recommendations for intervention(s) and an invitation for providers to engage federal and local governments in discussions of ways to unite families, schools, and communities in the battle against the costliest nutritional problem for children in the United States.

INTRODUCTION

Obesity and its co-morbidities currently account for over $250 billion per year in health care costs (~25% of total U.S. health care budget (1)) and is projected to increase to over $900 billion by the year 2030 (2). Obesity is a complex disease reflecting interactions of an increasingly permissive environment on a background of genetic predisposition and developmental programming (3-7). Results from 2017- March 2020 National Health and Nutrition Examination Survey (NHANES, Figure 1A) indicate that an estimated 21.5% of U.S. children and adolescents aged 2-19 years have obesity (Body mass index [BMI] > 95^th percentile for age and sex), an increase of 25% over the last decade, and 6.1% have severe obesity (BMI > 125% of 95th percentile for age and sex) (6,8,9). The prevalence of obesity is significantly higher in non-Hispanic Black and Hispanic children (Figure 1B). By adolescence, the prevalence increases to two-fold and nearly three-fold in these groups respectively compared to their non-Hispanic White or Asian counterparts.

Figure 1. Prevalence of obesity in youth. A. Trends in prevalence of obesity by age group in the past decade years (281). B. Obesity prevalence in youth by age, race, and Hispanic origin in United States, generated using data from National health and nutrition examination survey 2015-2018 (n=6710). Obesity was defined as BMI ≥ 95th percentile for youth 2-20 years of age using CDC 2000 growth charts and weight-for-height ≥ 97.7th percentile from birth to 2 years using WHO growth charts (282).

The prevalence of pediatric obesity and its co-morbidities, such as type 2 diabetes mellitus (T2DM), have been increasing in parallel. Pediatric obesity tracks into adulthood, especially if present in the peri-pubertal period (over 20-fold increased risk adult obesity) and if one or both parents have obesity (10). These problems disproportionately affect Black, Hispanic, and Native American communities (8,11,12). It is also worth considering that although a higher percentage of adults have obesity, the fractional magnitude of obesity prevalence among youth is growing faster. From 1971-2018 the prevalence of adults with obesity increased by about 2.8 fold (from about 15% to 42%) whereas the percentage of children with obesity increased by 3.8 fold (from about 5% to 19.7%) (8). Recent CDC reports using data from comprehensive electronic health records indicate that the monthly rate of BMI increase in children aged 2-19 years nearly doubled during the COVID pandemic period (0.100 versus 0.052 kg/m²/month; ratio = 1.93) and the prevalence of obesity increased from 19.3% in August of 2019 (pre-pandemic) to 22.4% in August of 2020. Children with higher BMI z-scores and between the ages of 6 and 11 years were most affected. (13).

Adults entering non-surgical weight loss treatment will typically lose weight for approximately 6-8 months followed by inexorable weight regain. Overall, only about 15% of adults with obesity are able to lose and sustain a greater than 10% weight loss, even with intensive lifestyle or pharmacological interventions. This number has not changed in over 20 years despite multiple new pharmacological and other treatment options (14-17). A key question is whether or not children are more responsive to interventions to treat, or to prevent, obesity.

Reviews of large lifestyle weight loss intervention studies indicate that children are more successful than adults in sustaining weight loss (usually defined by BMI z-score) provided that they remain involved in the intervention (see treatment discussions below) and that earlier intervention is more effective. Reinehr et al (18), performed retrospective quality assessments of 129 pediatric obesity programs at 6, 12, and 24 months and found that reduction of overweight was achieved by 83% of the children after 6 months, by 82% after 12 months, and by 76% after 24 months. The mean change of SDS-BMI was −0.20 ± 0.32 at 6 months, −0.19 ± 0.40 at 12 months, and −0.20 ± 0.54 at 24 months indicating an average of about 8% sustained reduction in adiposity. In adults with T2DM and overweight or obesity enrolled in the prospective LookAHEAD trial (15), the 1 year weight loss in the intensive lifestyle intervention group was 8.6± 0.1% (similar to children) but by year 2 it had fallen to 6.4±0.2% and by year 4 to 4.7±0.2% despite continued intervention.

Figure 2. Patterns of weight loss and regain in children and adults. A. On average, adults will lose weight for only about 6-9 months during lifestyle intervention to treat obesity. After this, most will then begin to regain weight (283-286). B. In contrast, children tend to lose more fatness (expressed as BMI z-score) and sustain their weight loss longer following a lifestyle intervention (61,62). This is especially true for younger children. *Based on DeJonge et al (286). †Based on Kraschnewski et al (16).

Meta-analyses of pediatric weight loss lifestyle interventions have generally noted reductions in BMI, BMI z score and weight that are influenced by both the type and duration of the intervention (19,20) and exceed results seen with lifestyle interventions in adults especially if intervention is initiated early. Figure 2 illustrates examples of patterns of weight loss followed by weight regain seen after lifestyle interventions in adults (Figure 2A) and children (Figure 2B). It is clear that most adults will lose a smaller fraction of total body fat and are less likely to sustain that loss than are children, and that younger children are more likely to reduce body fatness and keep it off than older children or adults. Similar patterns of weight loss and weight regain are seen in pharmacological interventions to treat adults with obesity.

These data indicate that there is a greater likelihood of successful treatment of obesity and reduced weight maintenance in children than adults but must be interpreted caution. Adult studies, such as LookAHEAD involve a continuous active intervention that may include medication while pediatric studies, such as Obeldisks (11) ⁽Figure 2) are only single year interventions with intermittent follow-up. Prospective pediatric studies such as Obeldisks may have a much higher attrition rate (about 70%) than adult studies such as LookAHEAD (about 10%), perhaps due to less contact with participants and type of intervention, as well as participant retention resulting in a smaller and less diverse study population of pediatric completers.

DEFINITION AND EPIDEMIOLOGY

The ideal diagnostic criteria for pediatric obesity would include some assessment of adiposity-related co-morbidity, the risk of persistence of the obesity into adulthood, as well as the risk of future morbidities that would be worsened by excess weight.

Several basic principles are pertinent to such an assessment:

During the first year of life there is an increase in weight for height followed by a decline and a second increase at about 6 years of age (designated as “adiposity rebound”). Early adiposity rebound, prior to 5 years of age, is associated with a higher risk of adult obesity (21,22).
The risk of persistence of pediatric obesity into adulthood increases with age, independent of the length of time that the child has been obese (3,23).
Growth patterns are familial and may be predictive of adult adiposity. A mildly overweight adolescent with a family history of adult obesity may be at greater risk for subsequent obesity than a severely overweight youth with a negative family history (3,23).
The risk of adiposity-related morbidity is strongly influenced by family history, regardless of obesity in the affected family members, and varies between racial/ethnic groups (3,23,24).

BMI is often used as a “surrogate” for body fatness. Although it does not measure body fat, it correlates with direct measures of body fatness within a population (25,26). In adults, obesity is frequently divided into categories– Class 1: BMI of 30-35 kg/m²; Class 2: BMI of 35-40 kg/m²; and Class 3: BMI ≥ 40 kg/m². Class 3 is also categorized as “severe” obesity. These definitions cannot be used in children because normative values for BMI are age- and sex-dependent (27). In 2007, the AAP Expert Committee recommended that children between the ages of 2-19 years with BMI > 95^th percentile are classified as “obese” and those with a BMI between the 85^th and 95^th percentile are classified as “overweight” (28) using the 2000 Centers for Disease Control (CDC) growth charts. These charts were constructed from data collected between 1963-1980, that included lambda-mu-sigma (LMS) parameters to calculate ten smoothed percentiles between 3^rd and 97^th percentile (28). However, extreme percentiles for heavier children extrapolated using CDC LMS parameters did not match well to the empirical data for the 99^th percentile obtained in the later years. Instead, a better fit to the empirical data was obtained by using 120% of the smoothed 95^th percentiles (29). This modification gave rise to the extended BMI growth charts that provides a flexible approach to describe and track children with obesity. (30). The American Health Association recommended classification of BMI ≥ 120% of 95^thpercentile as severe (equivalent to Class 2) obesity (31). Subsequent publications have defined overweight as BMI between 85-95^th percentile, Class 1 obesity as BMI between 95^th- 120% of 95^th percentile, Class 2 between 120% -140% of 95^th percentile and Class 3 as ≥ 140% of 95^th percentile, making the classification similar to that used in adults (32-34) (Figure 3). For children less than two years of age weight/recumbent length ≥ 97.7^th percentile based on the World Health Organization (WHO) charts is currently used to define obesity (24,35).

Figure 3. Normative BMI growth curves for boys and girls. Extended BMI curves for youth aged 2-20 years of age based on Gulati et al (30). Class 1 obesity defined as BMI between 95th- 120% of 95th percentile; Class 2 between 120%-140% of 9th percentile; Class 3 ≥ 140% of 95th percentiles (287).

Normative data have also been established for waist circumference during childhood (Figure 4). Waist circumference is measured at the level of the upper border of the right superior iliac crest with horizontal alignment of the measuring tape, parallel to the floor, lying snug, but not compressing the skin. These data are most helpful in identifying children at risk for insulin resistance, type 2 diabetes, and dyslipidemia. The limitation of waist circumference is in the difficulty in properly locating anatomic landmarks such as the umbilicus and superior iliac crest, especially in individuals with severe obesity and/or a large volume of subcutaneous adipose tissue.

As noted above, BMI does not directly measure body fat. Individuals at either extreme (low or high) of percent body fat may be incorrectly labeled solely based on BMI. In such cases, if the clinician is uncertain, further evaluation may require more precise methods of assessing body fat such as bioelectrical spectroscopy (BIS), air displacement plethysmography (BOD POD), dual-energy X-ray absorptiometry (DEXA) scanning, or Quantitative Magnetic Resonance (QMR) (36,37).

Obesity in childhood and adolescence predisposes to obesity in adulthood. In a meta-analysis of 200,777 subjects derived from fifteen prospective studies, Simmonds et al showed that youth with obesity were five times more likely to have obesity in adulthood. Over half of the individuals with obesity in childhood will have obesity in adolescence and nearly 80% of adolescents with obesity will continue to have obesity in adulthood (38). In a separate meta-analysis of thirty-seven studies, the same group showed that high childhood BMI was associated with an increased incidence of adult diabetes (OR 1.70, 95% CI 1.30-2.22), coronary heart disease (OR 1.20, 95% CI 1.10-1.31), and a range of obesity associated cancers (39). It should be noted that while childhood obesity persists when present, not all adults with obesity or its associated co-morbidities had obesity in childhood, re-emphasizing that obesity is a result of complex interaction between familial predisposition, likely from genetics, and the environment.

Figure 4. Waist circumference (measured at the iliac crest while subjects stood and placed their hands on opposite shoulders) curves for North American Children age 5-19 years derived from NHANES III data (196) by the Canadian Pediatric Endocrine Group (https://cpeg-gcep.net/content/waist-circumference-and-waist-height-ratio-charts). Charted indices for these variables at extreme of body fatness are currently not available.

In addition to initiating therapy in childhood when it is more likely to be effective, it is also important to identify the child who is “at-risk” of becoming an obese adult. The risk of adult obesity is higher in children with a first degree relative with obesity and also increases as the child approaches puberty. Whitaker et al (23), examined health records from 854 subjects born at a health maintenance organization in Washington State between 1965 and 1971 and tracked them into early adulthood (age 21-29 years). The odds ratio for a child with obesity (defined as BMI > 85%ile for age and sex) becoming an adult with obesity rose steadily from 1.3 at age 1-2 years, to 22.3 at age 10-14 years, and 17.5 at age 15 to 17 years of age. In contrast, the effects of parental obesity on odds ratio decreased with age from 3.2 at age 1-2 years to 2.2 at age 15-17 years. More studies like this using larger populations will be informative regarding predictors of having obesity in adulthood.

ENERGY HOMEOSTASIS

The first law of thermodynamics dictates that the accumulation of stored energy (fat) must be due to caloric intake more than energy expenditure. A sustained small excess of energy intake relative to expenditure will, over time, lead to a substantial increase in body weight. For example, a 50 kg individual who increases their daily caloric intake by 150 kcal (8 ounces of whole milk) above their usual daily energy expenditure (~1800 kcal/day) would gain approximately 8 pounds before sufficient fat-free mass (FFM) was reached to result in a new equilibrium between energy intake and expenditure (assuming approximately 30% of weight gain is FFM). This assumes, however, that there were no metabolic adaptations to maintain body energy stores in the face of overnutrition (40,41). In fact, adults maintain a relatively constant body weight, and most children tend to grow steadily along their respective weight percentile isobars for age, with little conscious effort to regulate energy intake or expenditure, despite the potentially large effects of small imbalances in energy intake versus expenditure.

The high rate of recidivism to previous levels of fatness by reduced-obese children and adults (42-47), and the tendency for individuals to maintain a relatively stable body weight over long periods of time despite variations in caloric intake (48), provide empirical evidence that body weight is regulated. It is now known that energy intake and expenditure are responsive to complex interlocking control mechanisms in which numerous afferent signals from the gastrointestinal, endocrine, central and peripheral nervous system, and adipose organs are ‘sensed’ by central nervous system tracts whose efferent systems affect energy intake and expenditure so as to maintain (or restore) weight (40,49). Adding to the complexity of this system’s interactions, the amount of energy stored in the body as fat also exerts potent effects on growth, pubescence, fertility, autonomic nervous system activity, and thyroid function, suggesting that humoral “signals” reflecting adipose tissue mass interact directly or indirectly with many neuroendocrine systems (40,50-54). Weight loss and maintenance of a reduced body weight are accompanied by changes in autonomic nervous system function (increased parasympathetic and decreased sympathetic nervous system tone), circulating concentrations of thyroid hormones (decreased triiodothyronine and thyroxine without a compensatory increase in TSH) (55-58), and appetite (increased hunger, reduced sense of fullness) (59) that are consistent with a homeostatic resistance to altered body weight, acting, in part, through effectors that mediate energy expenditure and intake.

Such a neurohumoral system to protect body energy stores would convey clear evolutionary advantages. During periods of undernutrition, the perceived reduction in energy stores would result in hyperphagia, hypometabolism, and decreased fertility (protecting females from the increased metabolic demands of pregnancy and lactation and the delivery of progeny into inhospitable environments). While carefully controlled studies of the effects of weight loss on energy expenditure in children are not yet available, the higher success rates in sustained fatness reduction in younger children versus adults discussed above suggests that these same systems appear to be more malleable in children prior to puberty (60-62).

MOLECULAR GENETICS OF BODY FATNESS

Heritability of Body Fatness

The storage of excess calories as fat would have been highly advantageous to our progenitors by increasing survival during periods of prolonged caloric restriction and conferring a reproductive advantage. The opportunities for our distant forebears to consume calories to the point of becoming morbidly obese and the likelihood of their survival to an age at which such co-morbidities as T2DM, hypertension, or hyperlipidemia were both low. Thus, it is likely through natural selection that the human genome would be enriched with genes favoring the storage of calories as adipose tissue (63,64). Conversely, there would be few, if any, evolutionary pressures to discourage obesity and ‘defend’ body thinness.

With the possible exceptions of the rare cases of obesity due to single gene mutations (see below) or specific anatomic/endocrine lesions (see above), body fatness is a quantitative trait reflecting the interaction of development and environment with genotype. Twin and adoption studies indicate that the heritability of body fatness and of body fat distribution in adulthood is 50 to 80%, [approximately equal to the heritability of height and greater than the heritability of schizophrenia (68%) or breast cancer (45%)] (65) (66). Studies have also identified significant genetic influences (heritability greater than 30%) on resting metabolic rate, feeding behavior, food preferences, and on changes in energy expenditure that occur in response to overfeeding (67-75). Genetic influences on resting energy expenditure (REE) are evidenced by studies demonstrating that African-American children tend to have lower REE than Caucasian-American children, even when adjusted for body composition, gender, age, and pubertal status (76).

The calculation of heritability in twin studies assumes that each member of a monozygotic or dizygotic pair is reared in the same environment, and that the degree to which body fatness is more similar within mono- than dizygotic twin pairs is due to the greater genetic similarity of identical vs. non-identical twins. Studies comparing adopted children with their adoptive and their biological parents assume that each child shares little or none of the immediate environment with each biological parent, and that the degree to which body fatness is more similar between children and their biologic vs. adoptive parents is due to the 50% of their genotype that each child shares with each biological parent. Based on twin studies, the heritability of body fatness appear to increase with age (77), illustrating the complex interactions of many obesity-risk allelic variants with the environment.

Common Single Gene Mutations Associated with Obesity

The pivotal role of genetics in the control of body weight is confirmed by the existence of rare single gene variants producing extreme obesity phenotype (e.g., Prader Willi, Bardet-Biedl, Alström, and Cohen syndromes). The most common monogenic cause of obesity – variants in MC4R – does not cause syndromic features, while others cause obesity in association with other distinctive dysmorphic phenotypes (67,78 ) (Table 1). The fact that mutations in different genes can produce obesity suggests that these genes may be part of a control system for the regulation of body weight, i.e., that feeding behavior and energy expenditure are integrated in a system with complex control mechanisms which can be disrupted at many loci. Further, the impact of a genetic change may not be a direct increase in weight – as an example – recent studies of Prader Willi Syndrome have demonstrated that the endocrine phenotype is due to a deficiency in prohormone convertase, an enzyme that has also been identified as a single gene mutation cause of obesity (79,80).

Table 1. Common Single Gene Mutations Associated with Obesity (67)
Syndrome/Gene	Chromosome	Phenotype
Alström syndrome/ ALMS1	2p14-p13 (Recessive)	Childhood blindness due to retinal degeneration, nerve deafness, acanthosis nigricans, chronic nephropathy, primary hypogonadism in males only, type II diabetes mellitus, infantile obesity which may diminish in adulthood.
Bardet-Biedl syndrome (22 different genes)	16q21 15q22-q23	Retinitis pigmentosa, mental retardation, polydactyly, hypothalamic hypogonadism, rarely glucose intolerance, deafness, or renal disease
Beckwith-Wiedemann syndrome	11p15.5 (Recessive)	Hyperinsulinemia, hypoglycemia, neonatal hemihypertrophy (Beckwith-Wiedemann Syndrome), intolerance of fasting
Börjeson-Forssmann-Lehman syndrome/ PHF6	X-linked	Intellectual disability, epilepsy, microcephaly, short stature, gynecomastia, hypogonadism, obesity, tapering fingers and short toes, multiple ophthalmological problems, coarse facial features, ptosis, large and long ears, supraorbital ridge
Carpenter /RAB23and MEGF8	Unknown (Recessive)	Mental retardation, acrocephaly, poly- or syndactyly, hypogonadism (males only)
Cohen /COH1	8q22-q23 (Recessive)	Mental retardation, microcephaly, short stature, dysmorphic facies
Leptin deficiency / LEP	7q31.3 (Recessive)	Hypometabolic rate, hyperphagia, pubertal delay, infertility, impaired glucose tolerance due to leptin deficiency.
Leptin Receptor / LEPR	1p31-p32 (Recessive)	Hypometabolic rate, hyperphagia, pubertal delay due to deranged leptin signal transduction.
Melanocortin 4 Receptor /MC4R	18q22 (Dominant)	Obesity – early onset hyperphagia, increased bone density
Neisidioblastosis	11p15.1 (Recessive or Dominant)	Hyperinsulinemia, hypoglycemia, intolerance of fasting
Prader Willi syndrome	15q11-q12 (Uniparental Maternal Disomy)	Short stature, small hands and feet, mental retardation, neonatal hypotonia, failure to thrive, cryptorchidism, almond-shaped eyes and fish-mouth
Pro-opiomelanocortin / POMC	2p23.3 (Recessive)	Red hair and hyperphagia due to low POMC production of alpha-MSH in hair follicles and the hypothalamus, respectively; adrenal insufficiency due to impaired POMC production of ACTH.
Prohormone Convertase/ PCSK1	5q15-q21 (Recessive)	Abnormal glucose homeostasis, hypogonadotropic hypogonadism, hypocortisolism, and elevated plasma proinsulin and POMC
Pseudohypo-parathyroidism (type IA, aka Albright’s) / GNAS	20q13.2 (Dominant)	Mental retardation, short stature, short metacarpals and metatarsals, short thick neck, round facies, subcutaneous calcifications, increased frequency of other endocrinopathies (hypothyroidism, hypogonadism)

Genome-Wide Association Studies (GWAS) of the Obesity Phenotype

The single gene mutations in humans listed in Table 1 are invariably associated with distinct phenotypes and marked (if not extreme) obesity. On the other hand, polygenic obesity may be phenotypically less extreme and with a more variable and subtle phenotype without any other syndromic features. GWAS of large populations have identified over 100 genetic loci as unequivocally associated with obesity-related traits (81-83) and over 500 loci associated with obesity-susceptibility (84), these allelic variants generally have been shown to exert only a small, but cumulative, effect on BMI (85).

In 2007, Frayling et al (86) reported a link between a SNP in the first intron of the FTO gene (rs9939609) and obesity in a GWAS of approximately 500,000 individuals with type 2 diabetes. Individuals homozygous for this SNP (AA) were approximately three kilograms heavier and at a 1.7-fold increased risk of obesity than those who were homozygous unaffected (TT). Since then numerous other FTO-related SNP’s have been identified that are associated with BMI (87,88). These SNP’s are especially relevant to the study of childhood obesity because of their frequency (14-18% AA; 39-50% AT; and 30-35% TT (89)) and the fact that the behavioral phenotype is evident in early childhood before obesity is manifest. Cecil et al. (90) used a three-pronged preload model to quantify energy intake in 4 to 10 year-old subjects genotyped with AA, AT, and TT alleles and found that the presence of an A allele was associated with increased energy intake and caloric density (kcal/gm) of foods chosen without any effect on energy expenditure (doubly labeled water method) or compensation index for increasing preload. Wardle et al. (89) reported that 4 to 5 year-old children who were homozygous (n=24) or heterozygous (n=66) for the FTO/FTM allele (AA or AT) and had eaten a meal to satiety, ate significantly more than control subjects (n=43, TT) when offered additional food, even when corrected for body fatness. The choice of snack was limited in this latter study and, thus, the authors were unable to comment on preference for calorically dense foods. Two separate studies of large cohorts (totaling over 36,000 individuals) reported no association of FTO genotype with increased BMI prior to the age of seven (86,91). There appears to be no effect of the A allele on energy output (88,92). Thus, behaviors that are premonitory of subsequent weight are evident and measurable in pre-obese children with allelic variants of FTO. These abnormal feeding patterns associated with increased energy intake (93) including decreased dietary restraint following a caloric preload (94,95), and ratings of hunger prior to or satiety after a meal (96) are not seen in already-overweight adults. These data emphasize the importance of studying eating behavior in subjects "at risk" for weight gain to understand the dynamics of food intake that favor the development of obesity.

More recently, use of polygenic risk scores (combination of the risk estimate apportioned by each common variant) using as many as 2.1 million common variants has enhanced the ability to quantify the susceptibility of obesity. Khera et al(4) used such a polygenic risk score in the 7,861 participants in the Avon Longitudinal Study of Parents and Children, a birth cohort recruited between 1991-92 and longitudinally followed to 18 years of age. The birth weight of the individuals in the top decile of the polygenic risk score was 0.06 kg (p=.02) higher compared to the bottom decile. By 8 years of age, the difference increased to 3.5 kg (p < .0001) and by 18 years, the difference in weight was 12.3 kg (p <.0001). The authors postulate that the aggregation of risk for obesity that can be conferred by having many common variants approaches the susceptibility equivalent to rare monogenic mutations in MC4R.

Epigenetics

The term “epigenetics” was first coined in 1942 by the British developmental biologist C.H. Waddington to refer to how gene regulation modulates development. In 1990, the molecular biologist Dr. Robin Holliday re-defined the term “epigenetics” as “the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms.” More recently this has been understood simply as the study of changes that affect the expression or “potency” of genes without necessarily affecting the nucleotide sequences of the genes themselves (97,98).

Epigenetics is extremely relevant to obesity in that it has allowed examination of the effects of the intrauterine environment, primarily in the form of factors affecting DNA methylation, histone acetylation, and expression of micro RNA’s, on gene expression relevant to obesity and its co-morbidities. Increased DNA methylation decreases the transcription of relevant genes and is affected by parental obesity, maternal diet (e.g., nutrition, folic acid content and other methyl donors), gestational diabetes (see below), and maternal medications (antibiotics and antipsychotics), smoking or exposure to chemicals such as bisphenol (99,100). Histones are proteins that “package” DNA into nucleosomes and post-translational modifications in the tails of histone affect the accessibility of DNA for methylation and translation. Loss of histone demethylase leads to obesity via decreased expression of PPARα and UCP1, and de-acetylation of the GLUT4 histone tail leads to impaired glucose transport (101,102). The human genome has been suggested to contain over 1000 micro (non-coding) RNAs (miRNAs), which may influence expression of more than 60% of mammalian genes by regulating gene expression. Each miRNA can interact with expression of multiple genes, including many involved in adipogenesis (103), that play pivotal roles in the development of obesity and its co-morbidities.

Major intrauterine environmental influences on the risk of subsequent obesity via these processes and others include maternal adiposity and gestational weight gain, under- and over- nutrition, gestational diabetes, maternal stress, and various chemicals, pharmaceuticals etc., to which the mother and fetus may be exposed during pregnancy.

Maternal weight impacts the fetus at multiple levels beyond those due to obesity risk alleles that may be inherited from either parent. This is exemplified by studies of offspring of mothers before and after bariatric surgery. The genotype of the mother is unchanged yet the fatness, blood pressure, circulating concentrations of insulin and gene expression relevant to diabetes, autoimmune disease, and vascular disease risk are all reduced in children who develop in the post-bariatric surgery intrauterine environment (104). Weight gain during pregnancy has a strong positive correlation with the incidence of large for gestational age babies and subsequent childhood obesity(105) augmented 2-5 fold in mothers with pre-partum obesity compared to those who were neither overweight nor obese prior to pregnancy.

Figure 5. U-shaped curve of odds risk for obesity at age 9-11 years based on birth weight. Curve is corrected for gestational diabets, gestional age, childs age, breast or formula feeding, highest level of parental education, sleep time, moderately vigorous physical acitivvity time (MVPA), sendentary time and healthy/unhealthy diet scores (288) weight Dashed lines identify 95% confidence intervals. Inserted text boxes indicate independent effects of small for gestational age (SGA) and large for gestational age (LGA) on various health parameters in adults.

Pre-Natal undernutrition (see Figure 5) reflects maternal undernutrition or compromised fuel delivery to the fetus–the latter usually due to placental dysfunction. Studies that have examined the prevalence of obesity in children conceived during periods of natural or man-made famine such as the Nazi-imposed Dutch famine of 1944-45 (the “Winter Hunger”) (106) report a small but statistically significant increase in the prevalence of obesity (defined as weight for height greater than 120% of WHO standards for 1948) in 19 year-old male military recruits whose mothers were malnourished only during the first trimester of pregnancy (2.77% prevalence if mother was in famine area vs. 1.45% if mother was outside of famine area during pregnancy) and a decrease in the prevalence of obesity among recruits whose mothers were malnourished during the child’s immediate post-natal period (0.82 % if mother was in famine area vs. 1.32% if mother was outside of famine area during pregnancy). It has been hypothesized that early intrauterine malnutrition might affect hypothalamic ("appetite center") development while the anti-obesity effects of early post-natal malnutrition might be due to suppression of adipocyte formation.

Long-term tracking studies of children who are small for gestational age, possibly due to prenatal undernutrition, have reported that, even when corrected for adult adiposity, birthweight is negatively correlated with the incidence of adiposity-related morbidities, including T2DM, hypertension, stroke, and cardiovascular disease, in adulthood (107-112). This association implies an interaction between the prenatal environment and development/function of pancreatic beta-cells and other organs such as the hypothalamus, liver, and kidneys that are involved in the regulation of adult energy homeostasis and cardiovascular function. As hypothesized by Barker (113-115), the metabolic, cardiovascular, and endocrine basis for adult adiposity-related morbidities may originate through adaptations that the fetus makes in response to undernourishment, especially when availability of calories in the environment that baby is born into is no longer limited. Therefore, the small-for-gestational-age baby should be considered to be at increased risk for adult morbidities that are exacerbated by increased adiposity (63).

Pre-Natal over nutrition (see Figure 5) is exemplified by the infant of a mother with gestational diabetes mellitus (GDM). The high ambient glucose concentrations of the prenatal environment stimulate fetal hyperinsulinemia, increased lipogenesis, and macrosomia. Since women with gestational diabetes are often overweight or obese, it is difficult to separate the metabolic effects of gestational diabetes on subsequent adiposity of offspring of mothers with GDM from the possibility that the mother has transmitted a genetic tendency towards obesity. Yet several studies have shown that GDM is associated with an increased risk of obesity in the offspring, independent of the degree of maternal obesity (116-119).

Maternal stress, which can be metabolic (e.g., obesity, diabetes, undernutrition, illness), psychiatric (e.g., depression, anxiety, bereavement), or pharmacological (e.g., steroids, antidepressants, antibiotics) have all been associated with increased risk of offspring obesity. These stressors affect developing neural systems regulating energy homeostasis, endocrine systems affecting risk of diabetes–including increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, immune system alterations resulting in increased circulating concentrations of pro-inflammatory cytokines, decreased concentrations of adiponectin relative to fat mass, and increased risk of hypertension (120,121).

Cocaine and marijuana: Exposure of the fetus to cocaine or marijuana during pregnancy has been reported to increase the likelihood of obesity in childhood and increase risk factors for T2DM (122-124). The mechanisms have yet to be ascertained.

Early Feeding Practices

For reasons discussed above, accurate assessment of the effects of early infant feeding practices on subsequent adiposity must control for possible effects of maternal adiposity as well as socioeconomic status and other factors that may affect the ability to breastfeed (125). Meta-analyses have shown that predominantly breastfeeding for at least 3-6 months is associated with significant reductions in the prevalence of obesity of their offspring through young adulthood (126-128), even when controlled for other adiposity-risk variables.

In addition to any benefits of the dietary macronutrient content on subsequent adiposity, observations suggest that the institution of a well-balanced diet in childhood may form the basis for long-term healthy dietary habits that will significantly lower adult cardiovascular disease risk even if the diet composition does not substantially affect weight (129). Studies have also identified positive correlations between the consumption of sugar-sweetened beverages, caloric density of snacks, fast food intake, the portion size of meals, and the hours of television watched (see below) with weight gain in children (129-135).

Early Life BMI Trajectories

A prospective longitudinal study of 7,738 U.S. children starting kindergarten in 1998-1999 showed that children with overweight and obesity at 5 years of age were four times as likely as their normal-weight counterparts to have obesity at 10-year follow-up. Among children who became obese between the ages of 5 and 14 years, nearly half had been overweight and 75% had been above the 70^th percentile for BMI at baseline, indicating that incident obesity for these children had occurred at younger ages (136). Many studies have examined the association of rapid weight, or weight/length or BMI trajectories in the first three years of life and noted association with overweight, obesity or severe obesity at 6 years to adolescence (137-141). One study simulated growth trajectories across life course using pooled data from nearly 42,000 children and adults representative of U.S. population in 2016 adjusting for secular trends. They estimated that a 2-year-old with obesity will continue a trajectory of rapid weight gain and has a 74.9% (95% CI 67.3 to 81.5) probability of being obese at age 35 years. These risks are higher for those with severe obesity.

Social determinants of health are the conditions in which people are born, grow, live, work, and age. These include the family, physical and social environment, each of which influences obesity either directly through children’s nutrition and activity or indirectly via added stress. Analysis of NHANES data has demonstrated a higher prevalence of obesity and severe obesity with greater age and lower education of the household head (142). This same study noted an association of severe obesity in youth residing in non-metropolitan statistical areas with more difficult access to large supermarkets. In a study of national sample of 3,748 children from US households receiving Supplemental Nutrition Assistance Program (SNAP) compared to those without, Gorski Findling et al noted higher odds of obesity amongst those receiving the SNAP program (OR 1.14 [95% CI 1.05-1.24]) and with access to a combination grocery/other store, compared to those with access to supermarkets with greater variety of fresh food. They also noted that in convenience stores, 26.1% of the average child’s total household food spending was on sugary beverages (SNAP 29.8% vs non-SNAP 15.5%) (143). In a study of households in New York City, Elbel et al noted that living farther than 0.025 mile (about half a city block) from the nearest fast-food restaurant was associated with lower rates of overweight and obesity, along with lower BMIz scores (144). The built and natural environments play a critical role in the access to physical activity (PA) for children. Street connectivity, defined as the directness of links and density of connection in street networks provides better access to outdoor PA such as walking, playing and cycling. Studies with perceived street connectivity by children, frequently near school, had higher odds of PA (OR 1.13, 95% CI 1.04-1.24). Similarly, higher odds of moderate to vigorous PA (OR 1.33, 95% CI 1.17-1.52) was noted with higher levels of street connectivity. No significant associations were identified with BMI or BMI z-scores (145). The same authors also conducted a metanalysis of natural environment with levels of PA in children. Ambient temperature was identified as the most consequential predictor associated with PA. An increase of 10 °F heating and cooling was associated with reduction in moderate to vigorous PA by 5 and 17 min respectively. No associations were reported with air quality (146).

Thus, a complex array of determinants contributes towards the risk of obesity and severe obesity in youth and will require a multipronged approach for intervention. (147-157)

Physical Activity, Sedentary Behavior, and Sleep

Behaviors related to PA and sedentary established in childhood have been shown to track well into adulthood(150,151) and are independent correlates of BMI and adiposity (158,159) (160,161). Meta-analyses of cross-sectional studies show negative associations of PA and positive associations of sedentary behavior (SB) with adiposity in children (162-164), that are further evident with direct objective (e.g., calorimetry) rather than subjective (self-reported) assessments (163). The implications of these findings for early intervention to treat and prevent pediatric obesity are discussed below.

MORBIDITIES ASSOCIATED WITH OBESITY IN CHILDREN

As in adulthood, obesity in childhood adversely affects every organ system (Table 2). Adiposity-related morbidities, such as hyperlipidemia, track well into adulthood (165) and pediatric obesity may be considered an independent risk factor for adult adiposity-related morbidities, even if the obesity does not persist (166). Certain morbidities, such as slipped capital femoral epiphyses, are the consequence of the biomechanical stresses associated with excess weight while others, especially cardiovascular morbidities, appear to be more closely related to central body fat distribution rather than absolute fat mass. The psychological stress of social stigmatization imposed on children with obesity may be just as damaging to some as the medical morbidities, resulting in significant body dissatisfaction, social anxiety, loneliness, and, especially in girls, somatic symptoms (167,168). These negative images of the obese are so strong that growth failure and pubertal delay have been reported in children due to self-imposed caloric restriction arising from fears of becoming obese (169).

Table 2. Pediatric Adiposity-Related Morbidities (165,167-173)
Cardiovascular	Hypertension, total cholesterol, low density lipoproteins, ¯ high density lipoproteins, metabolic syndrome
Respiratory	Abnormal respiratory muscle function and central respiratory regulation, difficulty with ventilation during surgery, lower arterial oxygenation, obstructive sleep apnea, asthma, more frequent and severe upper respiratory infections
Gastrointestinal	Nonalcoholic fatty liver disease, gallstones, gastroesophageal reflux disease
Endocrine	Type 2 diabetes, precocious puberty, polycystic ovarian syndrome, Vitamin D deficiency
Orthopedic	Coxa vara, slipped capital femoral epiphyses, Blount's disease, Legg-Calve-Perthe's disease, degenerative arthritis.
Dermatologic	Intertrigo, furunculosis, acanthosis nigricans (HAIR-AN Syndrome)
Immunologic	Impaired cell-mediated immunity, polymorphonuclear leukocyte killing capacity, lymphocyte generation of migration inhibiting factor, and maturation rates of monocytes into macrophages
Psychologic	Low self-esteem, anxiety, somatization, depression, eating disorders
Lymphatic	Obesity associated lymphedema of the lower legs
Malignancy	Higher lifetime risk of obesity related cancers

Pediatric Obesity and Cardiovascular Risk Factors

Obesity, hyperlipidemia, hypertension, and other risk factors for cardiovascular disease in children track well into adulthood (23,165,170-173). In long-term follow-up studies, adolescent fatness was a powerful predictor of mortality, cardiovascular disease, colorectal cancer, gout, and arthritis, irrespective of body fatness at the time that the morbidity was diagnosed (166,172). Therefore, it is possible that the metabolic groundwork for the chronic diseases of adulthood is laid down in childhood and the overweight youth must be assessed for both current adiposity-related morbidities and their future risk.

Pediatric Obesity and Type 2 Diabetes Mellitus

The incidence of youth-onset prediabetes and T2DM is increasing parallel with the rise in obesity in the US (174,175). Between 2001 and 2017, there was a 95.3% (95% CI 77.0-115.4%) relative increase in the prevalence of T2DM in youth < 19 years of age. The greatest absolute increase were observed among non-Hispanic Black and Hispanic youth (174). In the past 2 years of COVID-19 pandemic, the burden of youth onset T2DM has increased dramatically. In a review of two U.S. medical claims databases (~500,000 individuals), persons aged < 18 years with COVID-19 infection were more likely to receive a new diagnosis of diabetes (both Type 1 and Type 2) > 30 days after infection compared to those without or those with pre-pandemic acute respiratory illness (HR = 2,66 [95% CI 1.98-3.56]) (176). The underlying causes for this increase are yet to be identified.

Pathologic processes associated with diabetes, including the development of insulin resistance and deterioration of beta-cell function, progress more rapidly in youth-onset T2DM than in adult-onset disease. These factors result in worse glycemic control and an increased risk of early diabetes-related complications (177-179). In the 10-year follow-up of 500 youth with new-onset T2DM enrolled in the Treatment Options for Type 2 Diabetes and Adolescents and Youth (TODAY) clinical trial, the cumulative incidence of hypertension was 67.5%, dyslipidemia 51.6%, diabetic kidney disease 54.8%, nerve disease 32.4% and retinal disease 51.0%. At least one complication occurred in 60.1% of the participants, and at least two in 28.4%. Risk factors for the development of complications included minority race or ethnic group, hyperglycemia, hypertension, and dyslipidemia (180).

The pathophysiology of T2DM is discussed in the Endotext Diabetes section (181). Like obesity, T2DM is a complex metabolic disorder(182). In studies of adults and children with a strong family history of T2DM, it appears that impaired pancreatic islet-cell function is the first identifiable metabolic abnormality in some subjects who subsequently develop T2DM, while in other populations, insulin resistance is the first identifiable phenotype (183,184). These data, along with the observation that subjects may be insulin-resistant but not meet clinical definition for diabetes, and that many individuals with impaired β-cell function may not go on to develop T2DM (185,186), suggest that T2DM is due to a combination of insulin-resistance and an impaired β-cell ability to respond to that state of insulin-resistance. In this sense, a state of relative insulin resistance, or the expression of an underlying tendency towards conditions associated with insulin resistance, the major causes of which in adolescence would be pubertal hormonal changes and/or obesity, may act to “unmask” a pre-diabetic state of impaired insulin secretion in some individuals. Consistent with this, available evidence suggests that the incidence of T2DM in children peaks around puberty, as do the ethnic differences in the prevalence of pediatric obesity (187,188), coincidentally with the known decline in insulin-sensitivity and increase in adiposity in the peri-pubertal period (189-191).

Central body fat distribution, usually defined on the basis of waist circumference or the ratio of waist-to-hip circumference, is an independent predictor of adiposity-related insulin resistance in adolescents and adults (191-193)as well as other co-morbidity risk factors (194-196). There appear to be effects of ethnicity on the relative impact of body fat distribution on insulin sensitivity. In Caucasian-American children, increasing visceral adiposity is the best correlate of increased fasting insulin levels and insulin secretion during OGTT, and of glucose disposal during hyperinsulinemic-euglycemic clamp studies (191). In African American (but not Caucasian) pre-pubertal children, intra-abdominal adipose tissue volume was significantly correlated with fasting insulin concentrations and with insulin sensitivity as measured by area under the curve (AUC) during oral glucose tolerance testing (197-199). Other studies of African-American prepubertal girls have found that elevated fasting insulin concentrations and reduced insulin sensitivity are significantly correlated with greater subcutaneous, but not visceral, adipose tissue volumes (200). Because of the increasing frequency of T2DM among adolescents with obesity, and the worsening of diabetes-related morbidities that may result from delayed diagnosis, the clinician should be alert to the possible of T2DM in all adolescents with generalized and central obesity, and especially those with strong family histories of early-onset (< 40 years of age, one or more parent affected) T2DM (201).

ENDOCRINE CHANGES ASSOCIATED WITH OBESITY IN CHILDREN

The most common endocrine disorders associated with obesity are secondary to excess body fat and will correct with weight loss (Table 3).

Table 3. Endocrine Changes Associated with Obesity in Children (202-206)
Somatotroph	¯ basal and stimulated growth hormone release, normal concentration of insulin-like growth factor-I, accelerated linear growth and bone age
Lactotroph	basal serum prolactin but ¯ prolactin release in response to provocative stimuli
Gonadotroph	Early entrance into puberty with normal circulating gonadotropin concentrations may be due to earlier priming of the hypothalamic-pituitary-gonadal axis by estrogens created by aromatization of androgens in adipose tissue and/or by increased circulating concentrations of leptin associated with higher adipose tissue mass.
Thyroid	Normal serum T4 and reverse T3, normal or serum T3, ¯ TSH-stimulated T4 release resulting in TSH levels
Adrenal	Normal serum cortisol but cortisol production and excretion, early adrenarche, adrenal androgens and DHEA, normal serum catecholamines and 24-hour urinary catecholamine excretion
Gonad	¯ circulating gonadal androgens due to ¯ sex-hormone binding globulin, dysmenorrhea, dysfunctional uterine bleeding, polycystic ovarian syndrome
Pancreas	fasting plasma insulin, insulin and glucagon release, resistance to insulin-mediated glucose transport

There are, however, several endocrine or genetic syndromes in which obesity is part of a distinct symptom complex that often includes poor statural growth (e.g., hypercortisolism, hypothyroidism) (Table 4) and/or very distinct heritable phenotypes (e.g., Prader Willi; Bardet-Biedl syndromes) (Table 1). Assessment of skeletal maturation by bone age, and physical examination for age-appropriate secondary sexual characteristics as well as syndrome-specific morphology or symptomatology (e.g., hypotension, constipation in hypothyroidism, centripetal distribution of fat in hypercortisolism) can usually rule out these syndromes as causes of obesity.

Table 4. Other Diseases, Injuries, and Medications Associated with Obesity (67,206)
Disease	Structural/Biochemical Lesion	Clinical Features
Acquired hypothalamic lesions	Infectious (sarcoid, tuberculosis, arachnoiditis, encephalitis), vascular malformations, neoplasms, trauma, post-surgical	Adipocyte hypotrophy with little hyperplasia, headache and visual disturbance, hyperphagia, hypodipsia, hypersomnolence, convulsions, central hypogonadism-hypothyroidism-hypoadrenalism, diabetes insipidus, hyperprolactinemia, hyperinsulinism, type IV hyperlipidemia
Cushing’s Disease / Syndrome	Hypercortisolism	Moon facies, central obesity, ¯ lean body mass, glucose intolerance, short stature
Hypothyroidism	Hypothalamic, pituitary, or thyroidal	Hypometabolic state (constipation, anemia, hypotension, bradycardia, cold intolerance), cretinism (if congenital)
ROHHAD or ROHHADNET syndrome*	Hypothalamic	Hyperphagia, obesity, hypoventilation, adipsic hypernatremia, thermal dysregulation, GH deficiency, hyperprolactinemia,
Medications	Tricyclic antidepressants, Glucocorticoids, Antipsychotic drugs, Antiepileptic drugs, Sulfonylureas

*ROHHAD - rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation; ROHHADNET - rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation with neural crest tumors.

WEIGHT STIGMATIZATION (FAT SHAMING)

Weight stigmatization (devaluation and denigration of a person because of obesity) includes explicit and implicit weight bias and perpetuates the view that obesity and the difficulty in weight loss are the fault of the individual’s poor diet and exercise choices (207). Weight stigmatization is so common across age, gender, race, and ethnicity that it must be considered as a co-morbidity of overweight and obesity; is prevalent in children and adolescents regardless of their socioeconomic and demographic characteristics. Between 25% and 50% of children have been bullied and/or have been discriminated against based on their weight (167,168). Weight bias is reported among peers, families, teachers, health professionals, and multiple media outlets (207-209) and has been shown to precipitate unhealthy eating habits, psychosocial stress, and additional weight gain in children (209-211).

PREVENTION AND TREATMENT OF OBESITY: CLINICAL APPROACH TO THE PEDIATRIC PATIENT

Prenatal Care

Prevention of obesity in childhood includes early, including prenatal, identification of the child at risk for subsequent obesity and application of effective interventions to reduce that risk. Ideally, this process includes health professions involved in obstetrics, maternal-fetal medicine, and pediatrics.

Pregnancy-related modifiable risk factors for maternal under- and over- nutrition, SGA, LGA, pre- and post-natal rapid weight gain as well as childhood overweight and obesity include higher maternal pregravid adiposity, excessive gestational weight gain, gestational diabetes and hypertension, and smoking during pregnancy (212). Addressing any of these risk factors is beneficial to the health of the mother as well as the fetus. The likelihood of modifying these risk factors is variable. The Institute of Medicine (USA) recommends different ranges of weight gain for women who are underweight (12.5-18.0 kg if BMI < 18.5 kg/m²), have a BMI within the normal range (11.5-16.0 kg if BMI 18.5-24.9 kg/m²), are overweight (7.0-11.5 kg if BMI 25.0-29.9 kg/m²) or are obese (5.0-9.0 kg for BMI >30 kg/m²) (213). As discussed above, there are clear offspring-health benefits of maternal bariatric surgery (104). However, it is difficult to implement non-surgical weight loss plans in preparation for pregnancy and the health benefits of lifestyle interventions both before and during pregnancy on childhood adiposity and co-morbidities smaller and less sustained than observed with bariatric surgery (212,214). Benefits of better control of gestational diabetes are more substantial and persistent (215).

Initial Evaluation

Pediatric obesity is a persistent worldwide problem, and preventing pediatric obesity and its comorbidities is of paramount importance. The authors posit that every youth with overweight, obesity, and severe obesity should have an opportunity for medical management shared with the individual, the family, and the medical home. The 2017 Endocrine Society guidelines for pediatric obesity assessment, treatment, and prevention provide an excellent framework towards this goal (24) (Figure 6). A thorough medical and family history is crucial as in any chronic condition.

Figure 6. Algorithm for implementing the 2017 Endocrine Society guidelines on management of children and adolescents with obesity (24).

Initial, and subsequent, evaluations should include a dietary history of the child’s and family’s typical eating habits (including snacks and the frequency with which they consume sugar-added beverages and foods prepared outside of the home). A physical activity history should also be obtained, including school physical education, after-school activities, and activities of daily living (such as walking to school), family activities, and sedentary activities (such as television watching). The family history should encompass obesity, bariatric surgery, T2DM, gestational diabetes, and other comorbidities of obesity including sleep apnea and use of continuous positive airway pressure (CPAP).

A detailed physical examination focused on identifying possible causes of unwanted weight gain (e.g., enlarged thyroid gland, cushingoid body habitus) and weight–related co-morbidities (acanthosis nigricans, hypertension, etc. see Tables 2-4) should be performed. Laboratory studies should be guided by history and physical examination and at minimum include fasting measurements of glucose, lipids, hemoglobin A1c, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and vitamin D to screen for diabetes, dyslipidemia, fatty liver disease, and hypovitaminosis D. Because of the increased risk of polycystic ovarian syndrome (PCOS) in adolescents with obesity, total and free testosterone, as well as sex hormone binding globulin (SHBG), should be measured in girls with signs of hyperandrogenism, oligomenorrhea, or other symptoms suggestive of this disorder (216). Routine evaluation for endocrine etiologies of pediatric obesity are not recommended by the Endocrine Society (24) except if statural growth is compromised. The authors suggest amending this to include a broader list of phenotypes that may be related to thyroid disease. These should include any possible indices of hypothyroidism in the first 3 years of life to avoid the deleterious effects of early thyroid disease on subsequent development (217). They should also include screening for symptoms of acquired hypothyroidism, especially autoimmune thyroiditis and especially in adolescent females, with later onset weight gain or autoimmune disease (218).

Children under 5 years of age who are extremely obese, especially if they have concurrent adiposity-related morbidities, evidence of developmental delay, or other phenotypic features associated with the rare obesity syndromes (such as Prader Willi or Bardet Biedl syndrome) discussed above (Table 1), can be referred to a physician who specializes in the treatment and genetic evaluation of pediatric obesity (219). Targeted therapies are available for some of these conditions.

Health care providers are often confronted by the difficulty in deciding whether or not to attempt intervention in a child who is not obese but is overweight or has a growth trajectory that may be premonitory of obesity. Assessment of the risk of progression to obesity should be based on family history of growth patterns and of adiposity-related co-morbidities, any evidence of co-morbidity in the child, and familial readiness to engage in early intervention to prevent obesity. Parents of a child who is clearly “at-risk” maybe more reluctant to begin lifestyle or other therapies since the child is not obese. If parents are amenable then the same therapies used to treat obesity can be initiated. If the parents are not amenable then the health care provider should monitor growth of the child and co-morbidity risk and keep parents involved in the discussion of progression towards obesity and/or its co-morbidities.

Family based interventions are most effective for management of pediatric obesity (219-221). Efforts spent in assessing the home environment are critical to success of management. Factors such as parental and sibling adiposity, education, and the quality of the relationship between the primary caregiver and the child have all been identified as significant determinants of the likelihood of a successful pediatric weight loss intervention (222). The logical extension of these findings is that optimal therapeutic interventions must include support for the child’s family, regardless of the level of obesity of the family members (223).

The clinician should begin assessment of family therapeutic readiness by asking the entire family how concerned they are about the patient’s overweight, in a supportive manner designed to elicit cooperation from the family and patient. Examples might include asking, “Do you feel that weight is a problem?” or “What do you think that you could change to help you lose weight?” rather than, “Why can’t you control what you eat?” The discussion should emphasize the potential benefits of therapeutic intervention, including the importance of cooperation of all caregivers, the increased likelihood of diminishing adult body fatness with early adoption of consistent and long-term lifestyle intervention.

Treatment of the child with overweight or obesity must be individualized and the clinician should remain sensitive to issues such as ability of the parents to prepare meals for the patient, neighborhood safety or availability of adult supervision, which may impact on the availability of physical activity after school, and remain culturally sensitive in making dietary recommendations.

Therapeutic Intervention

The approach to management of a child with overweight or obesity is in many ways more complex than the same choice in an adult because of additional concerns regarding growth if negative energy balance is excessive. The major goal of management should be to diminish morbidity rather than to achieve a "cosmetically endorsed" body weight. While imperfect, BMI is clinically the most readily accessible parameter to assess the level of obesity. The “severity” of obesity should initially be assessed based on the BMI references provided above, presence of current morbidities such as T2DM, and risk of future adiposity-related morbidity (based on family history) (219). This increased risk of treatment-associated impairment of statural or brain growth is higher in younger children and caloric restriction to reduce weight should not be used in infants less than 2 years of age. Beginning therapy with the assumption that obesity is a choice and can be “fixed” easily by moving more and eating less is outdated and inaccurate in the current science of obesity and promotes weight stigmatization and “fat shaming”. Excessive emphasis on behavior and self-sufficiency may precipitate eating disorders, as well as other psychological disorders such as low self-esteem, anxiety, and depression – especially if long-term weight loss is unsuccessful, especially in the peri-pubertal stages (167). It is important to tailor the management for individual child and their family. Program adherence, defined as the number of contacts with the weight-management program, is a primary factor in successful weight loss for overweight children and adolescents (224). Maximizing adherence is like to include program modification over time in a given child. As more data accumulate regarding precision medicine approaches to identify genetic and other predictors of responses to different interventions, adherence and success are likely to improve. Clinicians can prescribe intensive, age-appropriate, culturally sensitive, family-centered lifestyle modifications (dietary, physical activity, behavioral) to promote a decrease in BMI (rather than weight). When weight is maintained at a constant level or weight gain is proportionally slower than height gain, BMI can reduce with increase in linear growth. In the otherwise healthy child with obesity with no evidence of co- morbidity, such modifications may be sufficient to maintain long-term health. In contrast, in a youth with severe obesity (BMI ≥ 120% of 95^th percentile of BMI) or presence of co-morbidity such as T2DM or hypertension, such management can be augmented with pharmacotherapy and/or bariatric surgery, as deemed suitable (225,226).

DIETARY RECOMMENDATIONS

The Dietary Guidelines for Americans, 2020-2025 can be used as a reference for dietary counseling (227). These guidelines emphasize following a healthy dietary pattern at every life stage with a focus on meeting food group needs with nutrient (and not calorie)-dense foods and beverages and stay within the appropriate calorie limits. Hence, the entire family can be engaged in culturally appropriate dietary modifications. Studies suggest that the long-term sustenance of such intervention is most successful with a supportive family. It is also important to convey to the family on the need for sustenance of such changes for long-term favorable outcomes. Encouragement can be provided by examining growth and growth velocity curves with patients and their families to illustrate progress. If appropriate, the significance of any evident reduction in morbidity (e.g., lowering of blood pressure or cholesterol) can be reinforced. Reasonable goals in the form of a "target" body weight at the next visit should be set at each office visit so that the patient and parents are aware of what is expected. These goals should be modest and attainable even if patients are only moderately compliant with their diet and exercise regimens since achievement of an interval “target weight" will also encourage the patient.

The caloric need of a person varies maintain, gain, or lose weight are dependent upon age, sex, height, weight and level of physical activity. The Dietary Guidelines provide estimated amounts of calories needed to maintain energy balance of various age and sex groups at three different levels of physical activity from toddlers to age 2 years, as well as ages 2 and older (227). These estimates are based on the Estimated Energy Requirements (EER) equations, using average reference height and weight by age and sex. These are a useful starting point to tailor the needs to that of the patient. It is useful to get an assessment of the current caloric intake from the families. However, self-reported caloric intake is often inaccurate. For direct assessment, the child's ad libitum diet can be observed and recorded by the parents for a minimum of five consecutive days. A diet diminished to 300 to 400 kcal/day below weight-maintenance requirements as assessed by dietary history or as calculated based upon formula relating anthropometry to energy expenditure, e.g., the Harris-Benedict Equation (228) should result in weight loss of approximately one pound per week. Note that since weight reduction per se causes decreased energy expenditure (both from decreased metabolic mass and whatever hypometabolic state is invoked by losing weight (40,49,67) and during weight loss, periodic downward adjustments of energy intake will be necessary to sustain ongoing weight reduction. The family should be instructed in long-term monitoring of caloric intake within, and outside of, the home and cautioned not to become overly critical or punitive towards the child if weight loss is slow or compliance is suboptimal.

The core elements of Dietary Guidelines for Americans sorted by food group are listed below:

Vegetables: Increased relative consumption of vegetables of all types – dark green; red and orange; beans, peas, and lentils; starchy; and other vegetables.
Fruits: Consumption of whole fruits rather than juices.
Grains: At least half of the consumed grains should be whole grains.
Dairy: Dairy intake should be focused on fat-free or low-fat milk, yogurt, and cheese, and/or lactose-free versions and fortified soy beverages and yoghurt as alternatives.
Protein: Protein intake should focus on lean meats, poultry, and eggs; seafood; beans, peas, and lentils; and nuts, seeds, and soy products.
Fats: Children need fats – both saturated fats and cholesterol for normal growth and brain development. On the other hand, trans fats, such as those from fried foods are unhealthy. Eggs, butter, whole dairy products and oils, including vegetable oils and those in seafood and nuts are recommended.

The guidelines also recommend limiting foods and beverages higher in added sugars (including those with high fructose corn syrup), saturated fat, and sodium. Less than 10% of calories per day should be derived from added sugars starting at age 2 years, and families should be advised to avoid beverages with any added sugars. In the US, 57-61% children derive > 10% of their energy from added sugars, 88% consume > 10% saturated fat and nearly 95% consume foods containing greater than the recommended sodium amount.(229-234) Simply reducing the consumption of these types of foods should in and of itself result in a net negative energy balance most likely by reducing hedonic “eating in the absence of hunger” (235,236) and other aspects of energy intake which have been found to be correlated with subsequent weight gain in children .

Ultra-processed (UPF) are defined as “Industrial formulations typically with 5 or more and usually many ingredients. Besides salt, sugar, oils, and fats, ingredients of UPF include food substances not commonly used in culinary preparations, such as hydrolyzed protein, modified starches, and hydrogenated or intensified oils, and additives who purpose is to imitate sensorial qualities of unprocessed or minimally processed foods and their culinary preparations or to disguise undesirable qualities of the final product, such as colorants, flavorings, non-sugar sweeteners, emulsifiers, humectants, sequestrants, bulking, de-foaming, anticaking, and glazing agents” (237,238). These “ready to eat” or “ready to heat” preparations are typically high in added sugar, trans-fat, sodium, and refined starch and low in fiber, protein, vitamins, and minerals. The consumption of UPF has increased by 20-50% per decade 2000-2015 in the USA, and to an even greater degree in low- and middle- income countries (239). Diets high in UPF is associated with adverse health outcomes including obesity, hypertension, dyslipidemia, diabetes and pre-diabetes in adults (240-243,244 ) and, more recently, in children (7); (245,246). Though the Dietary Guidelines for Americans has yet to issue recommendations regarding UPF consumption, we believe that the evidence that UPF’s promote obesity and many of its co-morbidities in children is more than sufficiently compelling to recommend avoiding them.

A helpful brochure to recommend healthy eating for children including Nutrition conversation starters can be obtained from https://www.dietaryguidelines.gov/professional-resources.

The composition of the diet should contain at least the minimal recommend amounts of protein, essential fatty acids, vitamins, and minerals. The 2017 consensus from the Endocrine Society (24) recommended the following basic principles of dietary intervention to achieve negative energy balance, which it should be noted would likely be beneficial to everyone regardless of adiposity:

Replace all sugary drinks (including juices, sodas, and whole milk) with water, noncaloric beverages, and low-fat or skim milk.
Create a balanced diet including vegetables, fruits, whole grains, nuts, fiber, lean meat, fish, and low-fat dairy products. Specifically encourage consumption of at least five servings of fruits and vegetables daily.
Reduce intake of calorie dense foods such as saturated fats, salty snacks, and high glycemic foods including candy, white bread, processed white rice, pasta, and potatoes.
Minimize consumption of foods outside of the home. Fast foods in particular.
Eat breakfast daily.

Based on available data it appears that dietary macronutrient composition in childhood does not significantly affect later adiposity (247) and that diets consisting of drastically altered proportions of nutrients may be dangerous and yield no better results than a limited intake of a nutritionally balanced diet (248,249). It should be noted that the results of these studies vary substantially and may be age-dependent. For example, in a retrospective study Davis et al (250)reported that synergistic effects between the duration of breastfeeding and low sugar-sweetened beverage intake in reducing the odds of obesity in toddlers who were Hispanic. In contrast, a recent study comparing the effects of the low fat versus low glycemic index diet in the treatment of obesity in a population of Hispanic American adolescents found no differences between groups based on dietary macronutrient composition (251) and a recent meta-analysis by Hall and Guo (252) found that low fat diets promoted greater fat loss than low carbohydrate diets in adults.

As noted above, nutritional counseling should encourage decreasing the use of calorically dense (high fat or high glycemic index) foods and adding more fruits and vegetables to the daily diet. The substitution of water for non-nutritious high calorie sugar containing drinks (juices, iced teas and soda pop) may be very helpful (225), at least transiently (253){Ebbeling, 2012 #10778}. In some cases, reductions in calorically dense foods and sugar-containing drinks through substitution and/or elimination alone can decrease calories and weight without changing the general pattern of food consumption in the family. When families eat at restaurants and fast-food vendors, they have less control over food choices than they do at home. Thus, reduction in the number of meals prepared outside the home may also be an effective weight-loss strategy. Parents and adult caregivers should understand the important role they play in the development of proper eating habits in their young children. The parents’ food preferences, the quantities and variety of foods in the home, the parents’ eating behavior and physical activity patterns all determine how supportive the home environment is to the child with obesity.

THERAPEUTIC EXERCISE

Physical activity may promote a slightly increased muscle mass, thereby raising total metabolic rate, and the putative effects of exercise to reduce visceral adipose tissue mass independently lower the risk of hyperlipidemia and diabetes mellitus (254-256). However, the energy cost of even vigorous exercise is low when compared to the caloric content of many "fast foods" or other "snacks", and exercise should not be viewed as a "license to eat". For example, walking at three miles per hour for one hour consumes about 200 kilocalories, about the same number of calories contained in a 1¾ ounce bag of potato chips. Use of "treats", such as ice cream, potato chips, etc., as incentives to exercise negates its impact. As with all interventions to reduce pediatric adiposity, increasing physical activity and decreasing sedentary behavior is most likely to be effective, sustained, and benefit the entire family if the entire family participates.

Combining the 2017 Endocrine Society statement on pediatric obesity (24) with other recommendations for physical activity in children (147), the following guidelines are suggested which again could be applied to the entire family, regardless of their adiposity:

Exercise should be fun, age-appropriate, and tailored to the child’s fitness level and ability and should involve large muscle groups (e.g., quadriceps) to increase energy expenditure. Exercise frequency, duration, and intensity should increase over time.
Moderate-to-vigorous physical activity should, on the average, encompass 90-120 minutes of the day in preschoolers and toddlers (usually unstructured physical activity) and at least 1 hour of the day in children 6 years or older (usually structured physical activity such as after school sports).
Improve sleep hygiene (10-13 hours per night for preschoolers and 8-10 hours per night for adolescents) in response to numerous studies demonstrating associations of decreased sleep duration and weight gain (257-259).
In order to address the issue of increased sedentary behavior due to screen time, the American Academy of Pediatrics provides a downloadable Family Media Plan in English and Spanish (healthychildren.org/MediaUsePlan) (260). This plan is for all children and can be personalized for every family depending on the children’s age(s), family priorities, time of the year (e.g., academic year versus vacation), etc., and includes elements such as screen free zones, screen free times, choosing good content, using medial together and digital privacy and safety. In its 2017 recommendations specifically for children with obesity, the Endocrine Society suggested that nonacademic screen time should be reduced to 1-2 hours per day and that other sedentary behaviors, such as digital activities, should be decreased (24).

While no specific aspect of the sedentary lifestyle has been shown to directly cause obesity, behaviors such as television viewing, reading, working at a computer, driving a car or commuting do exert effects on health. Television viewing appears to be directly associated with the incidence of obesity, and inversely associated with the remission of obesity. The impact of television viewing on obesity seems to be due to both displacing more vigorous activities and its effect on diet. Not only is television viewing a sedentary behavior, but food has also constituted the most heavily advertised product on children’s television in the United States. In Mexican-American children, adiposity was significantly correlated with time spent watching television but not with time spent watching videos (261), suggesting that the bulk of the positive association of television watching and adiposity is due to the approximately 60% of advertising that is devoted to food (134). Children and adolescents should be encouraged to view as little television as possible. Limitation of television, video games, and internet viewing will encourage greater participation in physical activity. Clinicians should encourage children to participate in organized or individual sports (participate, not watch from the bench) and advocate for better community- and school-based- activity programs.

If the patient is unable to lose weight and/or co-morbid conditions persist, consideration should be given to referral of the child to a physician specializing in the treatment of pediatric obesity. Weight-loss programs, weight-reduction camps, etc. are often not covered by medical insurance and should be considered for the child who is morbidly obese with some caution. Enrollment in a highly supervised environment may demonstrate to an overweight child that weight loss is possible and encourage them to continue. However, rapid weight loss may precipitate cholelithiasis (262) or eating disorders. A child may become overly pre-occupied with his/her weight and, even if a moderate degree of weight-loss is achieved, lose self-esteem. Obsession with weight on the part of the child or their family may lead to serious deterioration of intra-family relationships.

DIGITAL INTERVENTIONS

Technology based interventions provide a novel tool to add to the armamentarium for weight management in youth. Technologies can include information and communication technology, web-based interventions, mobile phone applications and smart-phone based interventions, text-messaging, and wearable technology. In a systematic review of 8 studies (n=582 youth) of technology-based interventions with or without wearable devices with a spread of intervention ranging from behavioral counseling via telehealth to text-message based reminders and family-based therapies, significant differences in BMI were reported by 5 of the 8 studies. Pooled analysis showed standardized mean difference of -0.61 (95% CI -1.10, -0.13, p <.01), albeit with significant heterogeneity. Interestingly, as is seen with in-person interventions, the effect was lower in the sub-group with parental involvement (263). Similarly, in a separate meta-analysis of 12 randomized controlled trials (3227 youth), use of wearable devices, such as pedometers or wristband activity trackers, had statistically significant reduction in BMI, BMI z-score and body fat, but not in waist circumference. The impact was higher in individuals with obesity compared to those with normal weight (for prevention of obesity) (264). Where accessible, such technologies can provide an additional tool for weight management in youth.

PHARMACOLOGICAL AND SURGICAL INTERVENTIONS

For youth with severe obesity or those with concomitant co-morbidity, both pharmacotherapy and surgical interventions can augment intensive lifestyle management prescribed above. Several pharmacological therapies have been approved by FDA for use in youth ≥ 12 years of age in the past 5 years and clinical trials with additional medications are ongoing at the time of this publication. Professional associations such as The Obesity Society, Pediatric Endocrine Society as well as other experts have provided guidelines for clinical considerations on the use of obesity pharmacotherapy(265-267). Figure 7 provides a mechanistic overview of pharmacotherapies

Figure 7. Mechanism of action of the available medications. Many of the currently used medications for obesity impact the centers for weight regulation in the brain including hypothalamus and the prefrontal cortex, as well as other organs. Abbreviations: NorEpi: norepinephrine; POMC: pro-opiomelanocortin; CART: cocaine- and amphetamine regulated transcript; AGRP: agouti-related polypetide; NPY: neuropeptide Y; GLP1R: glucagon like polypeptide receptor 1; LEPR: leptin receptor; GABA: gamma amino butyric acid; MC4R: melanocyte 4 receptor. Bupropion and Naltrexone are not approved for use in pediatrics for weight loss. The therapeutic preparation of leptin is called metreleptin. Figure created using biorender.com

Both the indications for pharmacotherapy and the available approved pharmacological interventions are different in children than in adults. General recommendations for use of pharmacotherapy include: a) availability of a multidisciplinary team including at least one pediatric specialist; b) severe obesity (BMI ≥ 120% of 95^th percentile or BMI ≥ 35 kg/m²) or presence of a co-morbidity with BMI ≥ 95^th percentile (or BMI ≥ 30 kg/m²); c) concomitant lifestyle intervention; d) continuation of medication(s) if there is ≥ 5% BMI reduction from baseline at 12 weeks on the optimal dose or arrest or slowing of weight gain; e) discontinuation if not tolerated or if dangerous side effects occur or persist despite dose adjustment (265). A list of available therapies and evidence for pediatric use are listed below with guidance on administration is provided in figure 8.

Figure 8. Pharmacotherapy for youth with obesity, approval status and available pediatric data.

Bariatric surgery is only approved in adolescents and, although the frequency of adolescent bariatric surgery is increasing, it still accounts for only about 1% of total U.S. bariatric surgery cases (268). Outcome studies of adolescent bariatric surgery have shown significant improvements in weight, cardiometabolic co-morbidity risk, and quality of life tempered a high incidence (57%) of hypoferritinemia and need for additional abdominal procedures (13%) (269). The American Society for Metabolic and Bariatric Surgery recommends the following selection criteria for adolescents eligible for bariatric surgery:

Body mass index ≥ 35 kg/m² and a severe comorbidity, with significant comorbidity with short-term effects on health or BMI 40 kg/m² or above with more minor comorbidities.
Physical maturity, defined as completing 95% of predicted adult stature based on bone age or reaching Tanner stage IV. This criterion is based on theoretical concerns that rapid weight loss might inhibit statural growth if an adolescent has not reached near adult height.
History of lifestyle efforts to lose weight through changes in diet and physical activity.
Ability and motivation of the patient and family to adhere to recommended treatments pre- and postoperatively, including vitamin and mineral supplementation.
Appropriate understanding of the risks and benefits of surgery on behalf of the adolescents
Supportive but not coercive family.

CONTRINDICATIONS TO BARIATRIC SURGERY INCLUDE:

Medically correctable cause of obesity
An ongoing substance abuse problem (within the preceding year).
A medical, psychiatric, psychosocial, or cognitive condition that prevents adherence to postoperative dietary and medication regimens or impairs decisional capacity.
Current or planned pregnancy within 12 to 18 months of the procedure.
Inability on the part of the patient or parent to comprehend the risks and benefits of the surgical procedure.

Both the American Society for Metabolic and Bariatric Surgery and the Endocrine Society have recommended that a multidisciplinary team consisting of a bariatric surgeon, a pediatrician specializing in obesity, a nutritionist, a mental health professional, an exercise physiologist, and a health care coordinator should be established to evaluate optimal therapy for a child who is a candidate for bariatric surgery based on the presence of co-morbidities and failure of other interventions.

ADDRESSING WEIGHT STIGMATIZATION

Health care providers have an opportunity to improve the quality of life and intervention outcomes for children with obesity by addressing weight bias (209). Recent specific recommendations include:

Avoid oversimplification: Recognize the multifactorial nature of obesity as a disease that may require long-term, or even lifelong, attention and challenge stereotypes that obesity, or the difficulty in losing weight, is a lifestyle choice rather than a biological issue.
Avoid weight bias: When speaking with the patient or their family focus on the chief complaint (even if it is not weight-related), feel free to discuss implicit and explicit weight bias with families, and support evidence-based care including medication or surgery.
Encourage a collaborative relationship: Ask if it is okay with the patient and family to discuss weight during an appointment, use person-first language (“having obesity” rather than “is obese”), acquaint them with the multifactorial complex nature of weight management, and explore alternative factors that contribute to higher BMI.

OTHER INTERVENTIONS

There are new types of intervention that are only recently being vetted in pediatric randomized clinical trials. Prebiotics, probiotics, and other manipulations of the gut microbiome have been suggested as possible means of treating or preventing pediatric obesity with some initial promising results in relatively small studies (270-272). There is a wide variability in the efficacy of school-based interventions but with more attention to the methodological differences between those that are more successful and those that are not, it may be possible to create a cost-effective practical means of addressing the burgeoning problem of pediatric obesity (273).

There are also a number of bills languishing in Washington that have been left in committee and not allowed to be aired for public debate. The Sugar-sweetened beverage excise task (SWEET) act, the Stop Subsidizing Childhood Obesity Act, and establishment of nutrition standards for all foods served and sold in schools have all been projected to return between 4 and 35 times the number of dollars invested in health care cost savings over the next 10 years (274). The failure of the SWEET Act, and other legislation that might affect childhood obesity rates, to get into open debate suggests that health care professionals dealing with the problem of pediatric obesity could be more vocal regardless of whether they support the legislation. Implementation of the improved school meals endorsed by the Healthy, Hunger-free Kids Act has been shown to result in significant improvement in school-meals and to be increasingly acceptable to students, with improvement in participation in school-based breakfast programs since its implementation (275,276). Any efforts to remove funding from the Healthy, Hunger-free, Kids Act (277) or the Supplemental Nutrition Assistance Program (SNAP), in particular SNAP-Ed, will potentially promote poor dietary habits and food insecurity (278-280) and should provoke a similar level of discussion by health professionals in public forums. These are important issues and commentary from those most familiar with the problem should be helpful in their evaluation.

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Behavioral Approaches to Obesity Management

ABSTRACT

Obesity is extremely prevalent, affecting 42.5% of people in the United States alone. Advisory panels recommend a 5-10% reduction in initial weight for adults with obesity, or for those who are overweight, with a weight-related comorbidity. This loss can significantly reduce the risk of developing type 2 diabetes and improve other cardiovascular disease (CVD) risk factors, as seen in the Diabetes Prevention Program and Look AHEAD trials. Greater reductions in weight produce even greater improvements in CVD risk factors. Weight loss can be achieved with a comprehensive lifestyle program that consists of dietary change, increased physical activity, and behavior therapy, provided in individual or group sessions. Behavioral treatment can be combined with diets of varying macronutrient composition as long as they induce a caloric deficit. Physical activity should be gradually increased over a period of 6 months, and although it is not effective as a stand-alone intervention for inducing a clinically meaningful mean weight loss, it is very important for facilitating weight maintenance and improving health outcomes. Principles of behavioral treatment include self-monitoring, stimulus control, and goal setting. Weight regain is common after an initial treatment period of 6-12 months, but frequent follow-up with an interventionist, which includes at least monthly counseling, can mitigate it. Treatments delivered by telephone, internet, or smartphone can be more easily disseminated to larger populations and can produce clinically meaningful mean weight losses if they include content similar to that of in-person lifestyle interventions and provide personalized feedback.

INTRODUCTION

Obesity, defined by a body mass index (BMI) ≥ 30 kg/m², is the most common nutritional disease in the United States, affecting 42.5% of adults (1) and 19% of children and adolescents (2). An additional 31% of American adults have a BMI in the overweight range of 25.0-29.9 kg/m². Obesity is associated with an increased risk of developing cardiovascular disease (3), hypertension, dyslipidemia, and type 2 diabetes mellitus (4), along with other clinical conditions including nonalcoholic fatty liver disease, gastroesophageal reflux, obstructive sleep apnea, and osteoarthritis (5-7). A weight loss of 5-10% of initial body weight improves these complications and has been recommended by expert panels sponsored by the World Health Organization (8), the National Institutes of Health (9), and several professional societies. Losses of this magnitude can be achieved with a high-intensity lifestyle intervention (also known as lifestyle modification or behavioral weight loss treatment), as described in the Guidelines for the Management of Overweight and Obesity in Adults (i.e., Obesity Guidelines) (10) developed by The American College of Cardiology, American Heart Association, and the Obesity Society.

Comprehensive lifestyle interventions include three key components: diet, physical activity, and behavior therapy. This chapter describes each intervention component and reviews the short-term and long-term effectiveness of this approach. Lifestyle interventions have traditionally been delivered in 30-90 minute, in-person, group or individual sessions by a trained interventionist (usually a registered dietitian, psychologist, exercise physiologist, or other health-care professional). Although this is by far the best-researched treatment modality, the past two decades have seen an exponential growth in digital and other remote treatment approaches, which are reviewed in the final section.

EFFICACY OF HIGH-INTENSITY, IN-PERSON LIFESTYLE INTERVENTION PROGRAMS

Interventions categorized as “high-intensity” by the Obesity Guidelines provide a minimum of 14 treatment sessions during the first 6 months (10). Maintenance sessions may be delivered at a reduced frequency thereafter. In trials conducted in academic medical centers, participants treated by a 1200-1500 kcal/day diet, combined with regular exercise and a comprehensive program of group or individual behavior modification, lose an average of 5-8% of initial weight in 6 months (9-11), and approximately 60-65% of patients lose ≥5% of their initial weight. The lifestyle programs provided in the Diabetes Prevention Program and the Look AHEAD study provide excellent examples of high-intensity interventions.

Diabetes Prevention Program

In the Diabetes Prevention Program (DPP), more than 3,200 participants with obesity or overweight and impaired glucose tolerance were randomly assigned to a placebo, metformin, or an intensive lifestyle intervention, with the goal of inducing a 7% weight loss in the latter group (12). Participants in the lifestyle intervention group were given 16 individual on-site counseling sessions with a registered dietitian in the first 24 weeks, followed by at least one contact every other month for the remainder of the study. They were prescribed a reduced-calorie, low-fat diet (1200-2000 kcal/day, depending on initial body weight), and 150 min/week of physical activity. After an average of 2.8 years, participants in the lifestyle intervention group lost a mean of 5.6 kg, compared to 0.1 and 2.1 kg in the placebo and metformin groups, respectively. The 5.6 kg weight loss translated to a 58% relative reduction in the risk of developing type 2 diabetes. Ten years after randomization, the lifestyle intervention group had regained most of their lost weight, but their incidence of type 2 diabetes remained 34% below that in the placebo group (13).

Look AHEAD (Action for Health in Diabetes) Study

The Look AHEAD study enrolled more than 5,100 individuals with overweight/obesity and type 2 diabetes mellitus, and participants were randomly assigned to a diabetes support and education (DSE) group or an intensive lifestyle intervention (ILI) group, with the aim of examining the long-term effects of a 7% weight loss on cardiovascular morbidity and mortality (14). Participants randomized to the DSE group received three group education sessions each year in the first 4 years, whereas participants in the ILI group received treatment similar to that in the DPP with some modification. During the first 6 months, ILI participants had 3 weekly group treatment sessions and one individual visit per month and replaced two meals per day with a liquid supplement (i.e., shake). They were instructed to consume 1200-1800 kcal/day (with calories adjusted based on initial weight). During months 7 to 12, ILI participants had two group sessions and one individual visit each month, and used meal replacements for one meal per day. For the next 3 years, participants were offered one individual on-site visit and one phone (or e-mail) contact per month.

After 1 year, ILI participants lost 8.6% of baseline weight, compared with 0.6% for the DSE group, and at year 4, mean weight losses were 4.7% versus 1.1%, respectively. These latter losses were maintained at 8 years, at which time patients in the ILI group lost 4.7% of initial weight, compared with 2.1% for DSE participants. The study was ended at a mean of 9.6 years of post-randomization follow-up because there were no differences in cardiovascular morbidity and mortality between groups. However, patients in ILI, compared to DSE, had significantly greater reductions in HbA_1C, lost more weight, had larger improvements in cardiovascular disease risk factors (i.e., reductions in systolic and diastolic blood pressure and levels of triglycerides), and used fewer diabetes, hypertension, and lipid-lowering medications. Analyses showed that the greater the weight loss, the greater the improvements in those risk factors (Figure 1) (15).

Figure 1. Change in risk factors by weight loss categories for the Look AHEAD cohort. Data in all figures are presented as least square means and 95% CIs adjusted for clinical sites, age, sex, race/ethnicity, baseline weight, baseline measurement of the outcome variable, and treatment group assignment. Figure is reprinted with permission from reference (15).

Compared to DSE, additional benefits in the ILI group included greater reduction of depression symptoms and remission or reduced severity of obstructive sleep apnea. The Look AHEAD and DPP studies both demonstrate that weight loss and long-term benefits to health can be achieved through participation in a lifestyle modification program. However, a follow-up assessment of ILI and DSE participants 16 years post-randomization continued to reveal no significant differences in CVD morbidity and mortality between the two groups (16).

LIFESTYLE INTERVENTION COMPONENTS

Dietary Recommendations

The primary goal of the dietary prescription in a behavioral weight loss program is to induce a 500-750 kcal/day deficit (10,11). For women, this involves consuming about 1200-1500 kcal/day, while for men the goal is about 1500-1800 kcal/day. Calorie targets also can be based on body weight, with 1200-1500 kcal/day recommended for people who weigh less than 250 lbs. at baseline and 1500-1800 kcal/day for those >250 lbs. (10,11). The ideal composition of dietary macronutrients for producing weight loss has been studied extensively, with options including low-glycemic index diets, Mediterranean-type diets, low-fat diets, and reduced-carbohydrate diets (17). A low glycemic index is based on eating a diet containing foods with a lower glycemic load, that are less likely to cause large increases in postprandial blood glucose levels (19,20). A Mediterranean diet focuses on consuming higher amounts of plant-based foods, including fruits, legumes, vegetables, monounsaturated fats such as olive oil, and fish; and reduced consumption of foods high in saturated fats, like red meat and butter (21). Low-fat diets provide 10% to 20% of calories from fat and recommend plant-based foods including whole-grains, fruits, and vegetables (22). A low carbohydrate diet approach, like an Atkins or “ketogenic” diet, is characterized by consuming as few as 20 g/day of carbohydrates, and focusing on foods that are higher in protein and fat (23).

The outcomes of comparative studies of these different types of diets have consistently concluded that adequate weight loss depends less on the macronutrient content of the diet and more on the caloric deficit (17). The POUNDS LOST trial supported this conclusion in a large, 2-year study that randomized patients to one of four diets with different macronutrient compositions, varying in proportions of fat, protein, and carbohydrate content (fat/protein/carbohydrate content: 20/15/65%; 20/25/55%; 40/15/45%; and 40/25/35%, respectively) (24). The study showed no difference in the amount of weight lost among the diet groups, all of which were designed to produce an energy deficit of approximately 750 kcal per day. Several other studies have also found that different dietary approaches produce weight losses that are comparable, provided there is a sufficient reduction in calories (25,26) (Figure 2). The use of portion-controlled diets have been shown to facilitate greater weight losses than diets of conventional foods, but this is primarily due to improved adherence to calorie goals and not to their macronutrient profile (27).

Figure 2. Change in body weight for participants in low-fat and low-carbohydrate diet groups after 24 months, based on random-effects linear model. Figure is reprinted with permission from reference (25).

Because it appears that caloric restriction contributes to weight loss more than the macronutrient composition of the diet, diets should be chosen based on patients’ personal preferences and by the presence of comorbid conditions. For example, Fabricatore et al. (28) demonstrated that a low-glycemic index diet produced greater improvements in HbA_1cin patients with overweight and type 2 diabetes than did a traditional low-fat diet, even though the two diets produced comparable weight losses. Low-fat diets appear to be associated with greater reductions in low-density lipoprotein cholesterol (24,25,29), compared to low-carbohydrate diets. The latter diets, by contrast, are associated with greater reductions in triglycerides (26,29-33), increases in high-density lipoprotein cholesterol (25,26,29-33), and improvements in HbA_1C in patients with type 2 diabetes (33). Table 1 summarizes the results of selected randomized trials that examined the effects of macronutrient composition on changes in weight and health outcomes.

Table 1. Weight Loss Results from Randomized Trials that Compared diets with Varying Macronutrient Compositions

Study

No. Lifestyle Sessions Provided

Dietary Intervention

Weight Change

Month

Comment/ Other Results

Dansinger et al⁽²⁶⁾

160 (51% F)

58% completed

Atkins (low-carb)

Zone (even distribution)

Weight Watchers (points based)

Ornish (low-fat)

-2.1 kg ^a

-3.2 kg ^a

-3.0 kg ^a

-3.3 kg ^a

All participants had hypertension, dyslipidemia, and/or fasting hyperglycemia.

Weight loss was associated with level of adherence.

Each diet decreased LDL/HDL ratio.

N No significant changes in blood pressure or blood glucose at 12 months in either group.

Das et al⁽³⁴⁾*

34 (% F unknown)

85% completed

Low-glycemic load

High-glycemic load

-7.8% ^a

-8.0% ^a

Triglycerides, total, HDL, and LDL cholesterol decreased in both groups.

Fabricatore et al⁽²⁸⁾

79 (80% F)

63% completed

Low-glycemic load

Low-fat

-4.5% ^a

-6.4% ^a

All participants had type 2 diabetes.

Larger reductions in HbA_1cin the low-glycemic load group.

Foster et al⁽²⁹⁾

63 (68% F)

59% completed

Low-carbohydrate (high protein, high fat)

Conventional (high-carbohydrate, low-fat)

-4.4% ^a

-2.5% ^a

HDL cholesterol increased more and triglycerides decreased more in the low-carbohydrate group.

Greater reductions in LDL and total cholesterol in the low-fat group at 3 months.

Foster et al⁽²⁵⁾

307 (68% F)

63% completed

Low-carbohydrate

Low-fat

-6.3 kg ^a

-7.4 kg ^a

HDL cholesterol increased more and triglycerides were lower only in the low-carbohydrate group.

Greater decrease in LDL at 3 and 6 months in the low-fat group.

Gardner et al⁽³⁰⁾

311 (100% F)

80% completed

Atkins (low-carbohydrate)

Zone (even distribution)

LEARN (calorie-restricted)

Ornish (low-fat)

-4.7 kg ^a

-1.6 kg ^b

-2.2 kg ^ab

-2.6 kg ^ab

HDL cholesterol increased more in Atkins than Ornish group. Triglyceride levels decreased more in Atkins than Zone group.

No differences in insulin or blood glucose between groups.

Systolic blood pressure decreased more in Atkins than in all other groups. Diastolic blood pressure decreased more in Atkins group than in Ornish group.

Sacks et al⁽²⁴⁾

811 (64% F)

7 9.5% completed

Low-fat, average protein (highest carbohydrate)

Low-fat, high-protein

High-fat, average-protein

High-fat, high-protein (lowest carbohydrate)

-3.0 kg ^a

-3.8 kg ^a

-3.2 kg ^a

-3.4 kg ^a

LDL cholesterol decreased more in lowest fat than in highest fat group. HDL cholesterol increased more with lowest carbohydrate than with the highest carbohydrate diet. All diets decreased triglyceride levels similarly.

All diets, except the highest carbohydrate, decreased fasting insulin (greater decrease in the high protein vs average protein diets).

Shai et al⁽³²⁾

322 (14% F)

8 4.6% completed

Low-fat

Mediterranean (moderate fat, restricted calorie with fat predominantly from olive oil and nuts)

Low-carbohydrate

-2.9 kg ^a

-4.4 kg ^b

-4.7 kg ^b

No significant change in LDL cholesterol in any group.

HDL cholesterol increased in all groups, significantly more in the low-carbohydrate than low-fat group.

Triglyceride levels decreased more in the low-carbohydrate than in the low-fat group.

In diabetic participants, only the Mediterranean diet group had a decrease in fasting glucose.

Insulin decreased in all groups, for both diabetic and non-diabetic participants.

All groups had a significant decrease in blood pressure.

Adiponectin levels increased, and leptin levels decreased, in all groups.

Stern et al⁽³³⁾

132 (17% F)

66% completed

Low-carbohydrate

Conventional (low-fat)

-5.1 kg ^a

-3.1 kg ^a

Triglyceride levels decreased more in the low-carbohydrate group than in the low-fat group.

HDL cholesterol decreased less in the low-carbohydrate group than in the low-fat group.

Changes in total and LDL cholesterol were not significantly different between groups.

Yancy et al⁽³⁵⁾

120 (76% F)

66% completed

Low-fat diet

Low-carbohydrate, ketogenic diet with nutritional supplements

-6.7% ^a

-12.9% ^b

All participants were hyperlipidemic.

Triglycerides decreased more and HDL cholesterol increased more in low-carbohydrate group.

Table is reprinted with permission from reference (18).

All studies were analyzed by use of an intention-to-treat population, with the exception as indicated by an asterisk (*).

Different letters (in superscript) indicate statistically significant differences in weight loss between groups.

F indicates female; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; HbA_1c, hemoglobin A_1c; MR, meal replacements; CVD, cardiovascular disease.

*A completer’s population was examined. †Results reported as “greater,” “larger,” “increased more,” etc. represent statistically significant differences between treatment conditions.

Physical Activity Recommendations

Physical activity is an important component of a comprehensive lifestyle intervention, in which participants are typically instructed to increase their physical activity gradually to approximately 150-180 min/week over the first 6 months. This goal can be achieved by engaging in moderate physical activity (e.g., brisk walking) for 30 minutes on 5 days per week (10,11,13). Physical activity can be increased by incorporating short bouts of lifestyle activity into individuals’ daily routines, such as increasing the amount of daily walking or using the stairs when possible, or by longer bouts of structured physical activity (e.g., at the gym). Individuals should be encouraged to engage in physical activities that they enjoy rather than be prescribed a particular activity. The recommended physical activity levels for facilitating long-term weight management are higher (225-300 min/week) than those for losing weight (36). The effects of physical activity on weight loss, the maintenance of weight loss, and CVD risk factors have been investigated extensively.

PHYSICAL ACTIVITY AND WEIGHT LOSS

Physical activity has a modest impact on weight loss when compared with the effect of caloric restriction (36). This was demonstrated in a 12-week study in which participants achieved losses of 0.3-0.6% (male vs female) of initial weight from physical activity alone, compared to 5.5-8.4% (female vs male) and 7.5-11.4% (female vs male) losses for participants who reduced their calorie intake and those who changed both diet and physical activity, respectively (37). The exercise performed in this study consisted of 30 min/day of moderate activity on 5 days per week. Similarly, Wing et al (38) reported weight losses of 2.1, 9.1, and 10.3 kg after 6 months in participants assigned to physical activity alone, diet alone, and diet plus physical activity groups, respectively, all of whom were provided behavioral intervention.

PHYSICAL ACTIVITY AND WEIGHT MAINTENANCE

Although exercise has a limited impact on weight loss during the initial phase of treatment, it plays an important role in weight loss maintenance. Several studies have shown that the more physical activity the patient engages in, the better the maintenance of lost weight (39,40). Jakicic et al (40), in a secondary analysis of a randomized controlled trial, demonstrated that women who exercised more than 200 min/week maintained greater weight losses than those who exercised 150-199 min/week or <150 min/week. Data from the National Weight Control Registry have also provided evidence that high levels of physical activity are characteristic of individuals who report long-term, sustained weight loss (41). The Registry follows patients who have lost a minimum of 13.6 kg (i.e., 30 lb.) in six months and maintained this loss for at least 1 year. Of these successful weight loss maintainers, 91% reported that they were exercising consistently, with women expending 2,545 kcal/week and men 3,293 kcal/week (42). Based on these findings and other evidence, the current recommendation by the American College of Sports Medicine is that, for weight maintenance, individuals should exercise at a minimum level equivalent to an hour of brisk walking per day (36).

PHYSICAL ACTIVVITY AND CARDIOVASCULAR HEALTH

Physical activity also is crucial for improving cardiovascular health for both individuals with obesity and those of average-weight. Even in the absence of significant weight loss, regular bouts of aerobic activity have been found to reduce blood pressure (43), lipids (44), and visceral fat (45), the latter of which is associated with improved glucose tolerance and insulin sensitivity in non-diabetic individuals and glycemic control in patients with type 2 diabetes (46,47). Several authors have evaluated the independent effects of cardiorespiratory fitness and adiposity on subsequent CVD mortality and have suggested that high levels of cardiorespiratory fitness significantly decrease the CVD mortality risk in individuals with overweight and obesity, regardless of adiposity. Barry et al (48) performed a meta-analysis of 10 studies and concluded that, compared to individuals who were fit and had normal weight, unfit individuals had twice the risk of all-cause mortality regardless of their BMI, whereas individuals who were fit and had obesity had similar mortality risks as normal-weight, fit individuals. Similarly, in a longitudinal study of 25,000 men, Lee et al (49) found that those who were lean but unfit had double the mortality rate of those who were fit and lean. These findings indicate that all individuals should increase their physical activity to improve their health, regardless of its impact on body weight.

Principles of Behavior Therapy

The third component of lifestyle intervention is behavior therapy, which refers to a set of principles and techniques used to help patients adopt dietary and activity recommendations. Behavioral principles were first applied to the treatment of obesity in the 1960’s and early 1970’s and, since then, have been developed into a program of behavioral and cognitive strategies (11). The core components of behavior therapy include goal setting, self-monitoring, stimulus control, and problem solving.

GOAL SETTING

In behavioral weight loss treatment, goal setting refers to setting specific targets for making changes to the patient’s calorie intake, physical activity, and eating and exercise habits (50, 51). Goals need to be objective and easily measurable in order to facilitate patients’ assessment of their progress. Patients are encouraged to have a target range for their total daily caloric intake (or other dietary goals), a daily or weekly exercise goal in minutes, and short- and long-term weight loss goals. Other behavioral goals are introduced as treatment proceeds. Patients should set goals that facilitate their losing about 0.5-1.0 kg per week, for a total loss of 5-10 percent of initial body weight at the end of the weight loss phase (at about 6 months). These goals should be trackable and should specify when and how the goal will be accomplished. During a typical treatment session, the lifestyle interventionist reviews each patient’s progress in achieving goals from the previous session and helps the patient set new goals. If the goals from a previous session are not met, the interventionist assists individuals with identifying and reducing barriers to goal achievement or with modifying their goals accordingly. In group programs, this information is often shared with the entire group to further increase accountability and support problem-solving.

SELF-MONITORING

Monitoring target behaviors in a systematic way is a crucial aspect of the behavioral approach to weight loss. Self-monitoring provides instant feedback about the effectiveness of behavior change efforts. It can answer the most important question about behaviors: are they getting better, staying the same, or getting worse? Daily records also function to increase patients’ awareness of target behaviors and their effect on weight change. Self-monitoring is strongly linked to success in weight loss. Individuals who regularly monitor their weight, activity levels, and eating patterns usually achieve the largest weight losses (52,53).

Patients are encouraged to record all foods and beverages consumed and their calorie content (or an alternative dietary target) to determine if they have met their dietary goals. A thorough self-monitoring report might also include the individual’s feelings that day, particularly those that were associated with excess or unplanned eating, or other individually-identified triggers for overeating. Tracking the minutes and type of physical activity or pedometer step counts can be used to monitor improvements in the patient’s activity level. Patients also should be instructed to weigh themselves regularly at home, at least once per week, and to keep a record of their weekly weights.

Although some patients prefer traditional paper records, the majority now track these targets using smartphone applications (apps) and other digital devices such as wearable physical activity trackers and “smart” scales that automatically record body weight. Although these digital tools increase self-monitoring consistency and are preferred by most patients (54), they have not been found to enhance weight loss when compared to traditional tracking methods (52, 55, 56). Novel tracking tools such as digital food photography and bite counting devices may further reduce the burden of active recording, but some studies have suggested that these methods are less strongly correlated with weight loss and may produce smaller mean losses than active recording methods (57).

In lifestyle intervention programs, patients review their self-monitoring records with an interventionist who helps them to assess their progress, set goals, and problem solve barriers to goal adherence. Individuals often underestimate calorie intake and overestimate physical activity (58), and interventionists can help patients who report meeting their calorie and activity goals but do not lose weight to identify additional sources of caloric intake. These may come from underestimates of portion sizes or hidden sources of fat and/or sugar intake. Interventionists also can help patients address barriers to effective self-monitoring, or set more flexible self-monitoring goals (e.g., record on fewer days per week), as appropriate.

STIMULUS CONTROL

The goal of stimulus control is to alter external and internal cues that influence eating and exercise behaviors (11, 50, 51). In classical conditioning, cues develop when two stimuli (e.g., objects, activities) are repeatedly experienced together, which creates an association between the two. The appearance of one stimulus can invoke the other stimulus. Food cues are cues that cause an individual to think about eating or about specific foods. These may include external cues, such as the sight or smell of food, or an activity that is frequently engaged in while eating, such as watching television. Internal food cues include both physical sensations and thoughts or emotions that the person has come to associate with eating. Similarly, activity cues include internal and external experiences that the person has come to associate either with being active (e.g., the sight of sneakers by the door) or being inactive (e.g., the couch).

Patients learn to reduce negative food and activity cues -- either by avoiding problem cues or by creating new habits in response to those cues -- and to enhance cues for desired behaviors. Examples of cue reduction include avoiding places that sell or serve high-calorie foods, staying away from all-you-can-eat buffets, and keeping any high-calorie foods that are associated with overeating out of the house. The patient can instead be encouraged to buy single portions of these foods on planned occasions. For cues that cannot be avoided, the patient may be encouraged to identify an appropriate alternative behavior, such as taking a 5-minute break instead of snacking when bored at work. To increase cues for healthy eating, patients can be taught to improve the visibility and availability of healthy, low-calorie foods in their home or workplace, such as by storing these foods at eye-level. They can also add cues that promote physical activity, such as arranging to walk at a certain time every day with a partner or leaving their gym bag in their car so that it is the first thing that they see when they leave work. By making these changes, patients can ensure that their work and home environments facilitate (rather than interfere with) weight loss.

COGNITIVE STRATEGIES

Strategies from cognitive-behavioral therapy (CBT) have been incorporated into many lifestyle interventions. CBT focuses on identifying, testing, and correcting maladaptive thoughts in order to change emotions or behavior. For example, thoughts like “I’ll never reach my weight loss goal; I might as well eat whatever I want.”) can reduce the likelihood that a patient will adhere to their dietary goals. Patients are taught to create a rational response to these negative thoughts, such as by noting that “My weight loss may be slower than I would like, but continuing to make healthy choices gives me the best chance of long-term success,” or by highlighting some of the benefits that they experience when they make healthier eating choices (50, 51).

Some lifestyle programs also have incorporated strategies from motivational interviewing that are designed to help patients resolve ambivalence about the costs and benefits of behavior change, identify reasons for change, and improve self-efficacy. More recently, alternative cognitive strategies derived from mindfulness and acceptance-based psychological treatments have been incorporated into weight loss interventions. These treatments promote non-judgmental, present-moment awareness and willingness to experience discomfort in order to pursue long-term goals rather than cognitive change. Thus far, programs that place a significant emphasis on any of these cognitive techniques have not consistently enhanced weight loss when compared to standard lifestyle interventions, and those shown to be superior have only increased weight loss by 1-2 kg (59). However, because fewer studies have incorporated these techniques into comprehensive, high-intensity lifestyle interventions, they remain promising targets for future research.

STRUCTURE OF IN-PERSON BEHAVIORAL TREATMENT: SHORT- AND LONG-TERM

The lifestyle interventions provided in studies like the DPP and Look AHEAD followed a structured curriculum that gradually introduced different behavior change skills. Detailed treatment descriptions can be obtained from the intervention manuals for these two studies (50, 51) or an adaptation of the DPP protocol provided by Wadden, Tsai, and Tronieri for in-person delivery in primary care settings (60). Behavioral weight loss interventions are most commonly delivered in group sessions, which have been found to be as effective as individual counseling for weight loss in several studies (61,62). It may be that any weight loss benefit of receiving personalized support with individual counseling is roughly equivalent to the benefits of a greater degree of social support, empathic understanding, and healthy competition among group members. However, group treatment is more cost effective than individual counseling.

Frequency and duration of contact during the weight loss period are additional predictors of weight loss success (10,61). This benefit is apparent in trials comparing high-intensity lifestyle intervention programs to programs that provided identical dietary and physical activity recommendations with a lower session frequency, as well as in systematic reviews and meta-analyses of the efficacy of lifestyle interventions. For example, in a study by Perri and colleagues (63) that compared three different visit schedules to a control condition, the group that received 8 treatment sessions in the first 6 months had a weight loss of 3.5 kg at month 24 that did not differ significantly from the 2.9 kg loss of the control group, whereas patients who received 16 sessions had a loss of 6.7 kg that was superior to both groups. Of note, the group that received 24 sessions in the first 6 month did not differ in weight loss from the 16-session group at any time, suggesting that there may not be a benefit of further increasing visit intensity (while increasing costs). In 2012, the United States Preventative Services Task Force recommended that weight loss programs include at least 12-26 intervention sessions per year for optimal weight loss (64). This recommendation was based on their systematic review, which reported weight losses of 4 to 7 kg for programs with that level of intensity compared to 1.5 to 4 kg in programs offering fewer than 12 sessions (61). These findings were consistent with the Obesity Guideline’s conclusion that programs that provided at least 14 sessions in the first 6 months produce a weight loss of 5 to 8 kg, those that provide 6-13 sessions (1-2 sessions per month) produce a 2 to 4 kg loss, and those that provide less than monthly sessions induce minimal weight loss (10).

For weight loss maintenance, frequent, long-term contact with an interventionist is the most successful method for preventing weight regain. Weight loss maintenance sessions are important for providing individuals with the support and motivation needed to continue with the behavior changes they have made, such as engaging in physical activity, eating a low-calorie diet, and self-monitoring. Wing et al (65) demonstrated that monthly in-person sessions were more effective in preventing weight regain over 18 months of intervention than was an education-control group or an internet-based intervention. Participants in the three groups regained an average of 2.5, 4.9, and 4.7 kg, respectively, after an initial weight loss of 19 kg.

Table 2. Recommended Components of a High-Intensity Comprehensive Lifestyle Intervention to Achieve and Maintain a 5-to-10% Reduction in Body Weight.*
Component	Weight Loss	Weight-loss Maintenance
Counseling	≥14 in-person counseling sessions (individual or group) with a trained interventionist during a 6-mo period; recommendations for similarly structured, comprehensive Web-based interventions, as well as evidence-based commercial programs	Monthly or more frequent in-person or telephone sessions for ≥1 yr. with a trained interventionist
Diet	Low-calorie diet (typically 1200–1500 kcal per day for women and 1500–1800 kcal per day for men), with macronutrient composition based on patient’s preferences and health status	Reduced-calorie diet, consistent with reduced body weight, with macronutrient composition based on patient’s preferences and health status
Physical activity	≥150 min per week of aerobic activity (e.g., brisk walking)	200–300 min per week of aerobic activity (e.g., brisk walking)
Behavioral therapy	Daily monitoring of food intake and physical activity, facilitated by paper diaries or smart-phone applications; weekly monitoring of weight; structured curriculum of behavioral change (e.g., DPP), including goal setting, problem solving, and stimulus control; regular feedback and support from a trained interventionist	Occasional or frequent monitoring of food intake and physical activity, as needed; weekly-to-daily monitoring of weight; curriculum of behavioral change, including problem solving, cognitive restructuring, and relapse prevention; regular feedback from a trained interventionist

*Data are from the Guidelines (2013) for the Management of Overweight and Obesity in Adults, reported by Jensen et al. (10). The guidelines concluded that a variety of dietary approaches that differ widely in macronutrient composition, including ad libitum approaches (in which a lower calorie intake is achieved by restriction or elimination of particular food groups or by the provision of prescribed foods), can lead to weight loss provided they induce an adequate energy deficit. The guidelines recommended that practitioners, in selecting a weight-loss diet, consider its potential contribution to the management of obesity-related coexisting disorders (e.g., type 2 diabetes and hypertension). The guidelines did not address the possible benefits of strength training, in addition to aerobic activity. DPP denotes Diabetes Prevention Program. Table is reprinted with permission from reference (66)

REMOTELY-DELIVERED LIFESTYLE MODIFICATION INTERVENTIONS

In-person interventions can be costly because they require adequate facilities for hosting the intervention, staff for checking in patients, and the time of trained providers to deliver the intervention. Travel time also can represent a cost and inconvenience for patients, and many individuals, particularly those in rural and economically disadvantaged urban areas, do not have adequate access to evidence-based care. Over the past two decades, a growing body of research has investigated the use of telephone, computer, and smartphone-based methods for delivering lifestyle interventions to patients. Larger numbers of individuals can be reached with these methods at a cost that is significantly less than that of in-person interventions, particularly if little to no provider input is required. The COVID-19 pandemic further highlighted the need to identify effective ways of delivering lifestyle interventions remotely, as in-person treatment programs were either suspended or quickly migrated to phone calls or videoconferencing platforms due to stay-at-home orders and social distancing policies.

Telehealth Delivery

Remote interventions delivered live by a provider via telephone or videoconferencing, often referred to as telehealth, produce weight loss outcomes that are most consistent with those of in-person interventions. This delivery method improves treatment access and reduces travel time and cost for participants, but it has minimal impact on provider time and training costs. Several large trials have compared individual or group telephone calls to in-person treatment delivery. For example, Donnelly et al (67) achieved median 26-week weight losses of 13.0% with group conference calls which did not differ from the 12.7% loss of patients who attended on-site groups (both also received a 12-week 1200-1500 kcal/day portion-controlled diet). Similarly, Appel et al (68) showed comparable weight losses at 24 months for participants who received telephone-delivered sessions compared to those that received in-person visits (4.6 kg and 5.1 kg, respectively). Telephone-based interventions also have shown to be effective for weight maintenance and appear to attenuate weight regain to a similar degree as ongoing in-person sessions (62,65,69).

In the past several years, videoconferencing platforms have become more widely accessible. These platforms provide the capability for remotely delivered face-to-face interactions, which allow for visual demonstrations and may enhance feelings of connection with the interventionist and/or group (70). This delivery format has yet to be compared to in-person intervention in a randomized trial; however, pilot and short-term studies report weight losses that are 3 to 8 kg larger than control or minimal intervention conditions (71-73), which suggests that videoconferencing also may produce weight losses that are similar in magnitude to those of in-person interventions.

Digital Delivery via the Internet or Smartphone

Digitally-delivered programs in which standardized intervention content is delivered via digitally-accessible articles, messages (e.g., e-mail or SMS), or pre-recorded videos further reduce costs and interventionist burden when compared to live interventionist delivery either in person or through telehealth. Some of the earliest interventions with digital session content were developed for delivery via the internet. In an early study, Tate el al. (74) demonstrated that an Internet-based behavioral approach consisting of email-based lessons, online self-monitoring of diet and physical activity, and e-mail feedback from an interventionist produced greater 6-month weight losses of 4.1 kg compared to the 1.6 kg loss achieved by participants who received an educational program (i.e., Internet resources with no specific instruction in changing eating and activity habits). As technology has evolved, digital programs have more typically been developed for mobile delivery via smartphone apps or in formats accessible via either the computer or smartphone. Intervention delivery via text message also has been evaluated, but typically produces small mean weight losses (1-2 kg) when used as a stand-alone intervention format (75).

Relatively few studies have directly compared the efficacy of digitally-delivered to in-person treatment. Harvey-Berino and colleagues (76) compared the same 24-session group lifestyle intervention delivered weekly: 1) in-person; 2) by internet (including online content, self-monitoring tools, and weekly chat groups); or 3) in a hybrid format (the internet program with monthly in-person meetings). Weight losses were 8.0, 5.5, and 6.0 kg, respectively, with in-person treatment superior to the other two groups. These findings, along with the results of multiple systematic reviews, suggest that the strongest digitally-delivered interventions produce short-term losses that are at least 20-35% smaller than those achieved with in-person counseling (77, 78). Such interventions are valuable given their wide reach and low cost, and the difference between the results of these digital interventions and in-person programs is likely to wane over time with regain. However, the average effect of digitally-delivered interventions is small (1-3 kg), highlighting the importance of identifying features associated with effective interventions (77, 78).

The provision of tailored feedback is by far the most commonly identified characteristic that differentiates effective from less effective digital interventions (77, 78). In earlier digital trials, feedback was provided directly by an interventionist. Increasingly, digital programs provide fully-automated, personalized feedback, generated from algorithms that analyze participants’ self-monitoring data. This tailored automated feedback appears to produce weight losses that are similar in magnitude to programs with interventionist-delivered feedback (e.g., 79, 80). A 2015 study by Martin and colleagues (81) evaluated a combined approach providing participants with highly personalized automated and weekly interventionist-initiated feedback (by phone, email, or app), in addition to app-based lesson materials, in an effort to maximize weight loss. Participants were given activity monitors and smart scales, and the app delivered automated graphic feedback comparing their physical activity and weight loss to expected targets (calculated based on their starting weight and calorie prescription). If participants’ weight losses fell outside of the expected range, they were prompted to select a behavioral strategy (e.g., use portion-controlled foods) to get back on track. In this 12-week pilot study, intervention participants lost 9.4 kg compared to 0.6 kg in the control group (81).Additional research is investigating the potential for just-in-time adaptive interventions (JITAIs) that use machine learning to identify individual risk factors for behavioral lapses and provide tailored feedback and intervention strategies at the times when an individual is most at risk. An initial evaluation of a JITAI intervention that was designed to promote dietary adherence by predicting dietary lapses produced a 10-week weight loss of 4.7% when combined with an app-based commercial weight loss program (82).

User engagement has been found to correlate with weight loss in several digital trials, making it another potential target for improving the efficacy of digital interventions. One approach for enhancing engagement is to increase the interactive quality of the digital program. Thomas, Leahey, and Wing (83) tested the efficacy of a 12-week online program that provided interactive lessons that incorporated videos, quizzes, and practical exercises. The program also provided self-monitoring tools and fully automated weekly feedback based on participants’ recorded data. At 6 months, intervention participants lost 5.4 kg, compared to 1.3 kg for control participants who received static lessons about the benefits of weight loss (without behavioral strategies). Other efforts to increase interactive engagement have incorporated lifestyle programs into social media platforms, virtual reality, or online games, and several of these interventions also have produced mean weight losses of 4-5 kg (84). A recent study by Vaz and colleagues (85) combined several of these techniques into a smartphone app that provided automated feedback on weight and physical activity recorded via smart scale and activity tracker, respectively; text- and app-initiated engagement prompts from an interventionist; social networking and sharing of food and exercise data; and peer competitions based on dietary and physical activity adherence. The app produced a mean weight loss of 7.2 kg at 6 months, which was 4.2 kg larger than a control group that received two weight management visits.

Unfortunately, the efficacy of most commercially-available weight loss apps has not been systematically evaluated. A majority of these apps include only a small percentage of the behavioral strategies typically featured in intensive lifestyle programs (86), and most do not provide tailored feedback. Such programs are not likely to induce a clinically meaningful weight loss for most individuals. For example, the highly popular app, MyFitnessPal, which helps users set a calorie goal and track food intake, produced a mean loss of only 0.03 kg in 6 months in primary care patients, compared with a gain of 0.3 kg in controls (87). The frequency of logins declined sharply after the first month (to close to 0), which again underscores the problem of maintaining user engagement with digitally-delivered interventions that do not provide interactive content. Results have been more promising for online and app-based commercial programs that do provide comprehensive intervention content. Weight losses of 4-5 kg were achieved at 3-6 months in a randomized trial evaluating an online commercial program that provided nine weekly e-mail delivered video lessons, online content (e.g., recipes), self-monitoring tools, personalized summaries of self-monitoring data, and the option to chat with an interventionist online (88). Overall, these findings suggest that providers can support their patients’ weight loss by helping them to identify digital programs that offer comprehensive session content and personalized feedback.

CONCLUSION

There is clear evidence that intensive lifestyle interventions are effective in helping patients with obesity to lose 5-10% of initial body weight, a loss that is associated with improvements in CVD risk factors and other obesity-related comorbidities. Lifestyle approaches emphasize prescriptions for dietary intake, increased physical activity, and behavioral skills such as self-monitoring. Traditionally, these interventions have been delivered in-person by a trained interventionist, which limits their potential dissemination. It is also possible to achieve a clinically meaningful weight loss with digitally-delivered programs that include little to no contact from an interventionist, provided the intervention provides comprehensive session content, tailored feedback, and features that promote user engagement.

One of the most challenging aspects of behavioral weight control is keeping off lost weight. Several strategies can facilitate this goal, including maintaining patient-provider contact beyond the initial weight loss intervention, either in-person or remotely, and prescribing high levels of physical activity after weight is lost in the first 6 months. In addition, the more that patients practice the skills used by participants in the National Weight Control Registry, the more likely they will be to maintain their weight loss.

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Islet Transplantation

ABSTRACT

Transplanting islets of Langerhans consists of implantation in the recipient’s hepatic portal system of endocrine pancreatic tissue, with a variable degree of purification. The field of islet transplantation has evolved significantly since the initial attempts by doctors Minkowski and von Mering in 1882, with remarkable acceleration over the last four decades, thanks to the incredible efforts of the research community worldwide, with continuous improvements in cell processing and transplantation techniques, patient management and development of specific immunotherapy protocols. Restoration of beta-cell function can be obtained by transplantation of allogeneic islets in both non-uremic (Islet Transplant Alone, ITA) and uremic (Simultaneous Islet and Kidney, SIK and Islet After Kidney, IAK) patients with diabetes, providing long-term sustained function and improved metabolic control even when requiring exogenous insulin (i.e., suboptimal islet mass transplanted or development of graft dysfunction). Preservation of beta-cell function is now attained in virtually all recipients of islet autografts, a therapeutic option that should be considered for individuals undergoing total pancreatectomy for non-malignant conditions and, as recently reported for selected cases with malignant conditions. In addition, islet transplantation represents an excellent platform toward the development of cellular therapies aimed at the restoration of beta-cell function using stem cells in the near future. In this chapter, we will review the state-of-the art of clinical islet transplantation.

INTRODUCTION

Diabetes affects 537 million adults (20-79 years) throughout the world (2021) and this number will rise to 643 million by 2030 and 783 million by 2045 (IDF Diabetes Atlas 10th edition, https://diabetesatlas.org/). Many cases of diabetes are successfully treated with life-long multiple daily injections of exogenous insulin and monitoring of blood glucose levels. In the last decades significant improvements in insulin therapy thanks to new preparations (i.e., ultrafast and long-lasting insulin analogues) and the adoption of intensive diabetes management (infusion pumps and continuous glucose monitoring system) have resulted in an overall improvement of patients’ glycemic control and a decreased incidence of chronic complications of diabetes (1,2). However, exogenous insulin administration cannot attain the desirable tight control in the majority of diabetics (3-5), cannot avoid the long-term complications of diabetes in all patients and the life expectancy of patients with diabetes is still shorter compared to that of the general population (6-8). A broad international assessment of treatment outcomes in children and adults with T1D (including 324,501 people from 19 countries in Australasia, Europe and North America) showed that the proportion of patients with HbA1c <7.5% (58 mmol/mol) varied from 15.7% to 46.4% among 44,058 people aged < 15 years, from 8.9% to 49.5% among 50,766 people aged 15-24 years and from 20.5% to 53.6% among 229 677 people aged ≥ 25 years (9). Diabetes is one of the leading causes of end-stage renal disease, blindness and amputation (10). In principle, the treatment for type 1 diabetes, type 3c diabetes and many cases of type 2 diabetes lies in the possibility of replacing destroyed or exhausted beta cell mass in order to restore two essential functions: sensing blood sugar levels and secreting appropriate amounts of insulin in the vascular bed, ideally into the portal system. Currently, the only available clinical approach of restoring beta cell mass in patients with diabetes is the allogenic/autologous transplantation of beta cells (i.e., pancreas or islet transplantation). Clinical trials performed in the last three decades have shown that restoration of beta-cell function via transplantation of isolated islet cells or vascularized pancreas allows reproducibly achievement of a more physiological release of endocrine hormones than exogenous insulin in subjects with diabetes (11). Transplanting islets of Langerhans consists of implantation in the recipient’s hepatic portal system of endocrine pancreatic tissue, with a variable degree of purification. Isolated islets are transplanted using minimally invasive techniques with lower morbidity than vascularized pancreas transplantation, which requires major surgery. The field of islet transplantation has evolved significantly since the initial attempts by doctors Minkowski andvon Mering in 1882 (12), with remarkable acceleration over the last three decades, thanks to the incredible efforts of the research community worldwide, with continuous improvements in cell processing and transplantation techniques, patient management and development of specific immunotherapy protocols. In addition, islet transplantation represents an excellent platform toward the development of cellular therapies aimed at the restoration of beta-cell function using stem cells in the near future. In this chapter, we will review the state-of-the art of clinical islet transplantation.

WHEN TO CONSIDER ISLET TRANSPLANTATION?

Transplantation of pancreatic islet may be considered as a therapeutic option in several conditions associated with loss of beta-cell function (Table 1). The procedure may be performed as Islet Transplant Alone (ITA) in non-uremic subjects, an option generally indicated for the treatment of iatrogenic (surgery-induced) diabetes and for non-uremic patients with Type 1 Diabetes. Subjects with end-stage renal disease (ESRD) may be considered for Simultaneous Islet-Kidney (SIK) or, if already undergone renal transplantation, Islet After Kidney (IAK) transplantation, respectively. In special situations, transplantation of islets may be considered in combination with other organs (i.e., in the context of multi-visceral transplantation following exenteration comprising the pancreas) (13).

The source of the islets for transplantation may be the patient’s own pancreas (autologous or auto-transplant) mainly when surgical removal of the gland is required due to different conditions. After total pancreatectomy, the subject develops surgery-induced (iatrogenic) insulin-requiring diabetes. Introduced in the early 1970’s (14), islet auto-transplantation allows achieving optimal metabolic control without the need for exogenous insulin in approximately 70% of the cases when adequate islet numbers can be recovered from the pancreas (generally >250,000 islet equivalents). More than 500 auto-transplant in patients with near-total or total pancreatectomy have been performed to date (15). The largest series were published by the University of Minnesota (16-19), the University of Cincinnati (20,21), and Leicester (22-25). Even when an inadequate islet mass to attain insulin-independence has been recovered, stable metabolic control and excellent management can be achieved in most subjects undergoing autologous islet transplantation (18,26-31). Islet auto-transplantation is currently reimbursed by health insurance in the United States. In the past auto-transplant has been performed almost exclusively in patients undergoing pancreatectomy because of chronic pancreatitis, successfully preserving β-cell mass and preventing diabetes after major pancreatic resections (15,16,32,33). Additional indications for auto-transplant other than chronic pancreatitis are still controversial (34), and have been limited to the procedure performed only in small case series (35-40) of benign enucleable tumors or pancreatic trauma. Recently, broader selection criteria for auto-transplant were published (39,41), exploring the possibility of extending auto-transplant to patients with known malignancy, either having completion pancreatectomy as treatment for severe pancreatic fistulae or extensive distal pancreatectomy for neoplasms of the pancreatic neck or pancreatoduodenectomy because at high risk of pancreatic fistula (Table 1). Of note, a randomized, open-label, controlled, bicentric trial (NCT01346098) aimed to compare pancreaticoduodenectomy (PD) and Total Pancreatectomy with Islet AutoTransplantation (TP-IAT) in patients at high risk of Post-Operative Pancreatic Fistula (POPF) was recently published. The results indicate that TP-IAT can be considered a valid alternative to PD in these patients, as it reduced complication number, severity and length of hospital stay. Of note, a trend toward a reduction of mortality, even for patients with malignancy was also evident. As expected, TPIAT was associated to a higher risk of diabetes, but IAT was able to preserve, at least in part, the endogenous insulin secretion, mitigating the impact of the pancreoprivic diabetes and assuring a good metabolic control without severe hypoglycemic episodes. In the field of islet transplantation, this study definitively confirmed IAT could be indicated for pancreas diseases other than chronic pancreatitis, suggesting the possibility to extend IAT indications (Milan protocol(42)). For the first time in a randomized prospective design, it was confirmed that IAT is feasible, safe and effective in patients with periampullary cancer, in agreement with previous series of patients undergoing IAT after pancreatic resection for a wide spectrum of disease besides chronic pancreatitis (43) (44) (37). This approach will be tested in further studies in the next years, such as the recently started TPIAT-01 trial (NCT05116072), which hypothesize that TPIAT rather than PD may improve the access to adjuvant chemotherapy in patients with adenocarcinoma.

In the case of subjects who lost islet function (mainly patients with Type 1 Diabetes or, more rarely, previous total pancreatectomy) the only option currently available for transplantable islet cells is allogeneic donor pancreata. These are generally obtained through multi-organ donation after cerebral death, following conventional donor:recipient ABO blood type matching. The use of a segment of the pancreas from living-related donors is technically feasible (45,46), but at the present time not preferred for islet transplantation due to the limited duration of graft function after transplantation of suboptimal islet numbers under standard immunosuppressive protocols, as well as the intrinsic risks for the donor (i.e., morbidity and risk to develop diabetes)(47).

Table 1. Indication for Islet Transplantation
Condition	Procedure	Type of Transplant
*Diabetes Mellitus*
Type 1	ITA, SIK, IAK	Allogeneic
Type 2	ITA, SIK, IAK	Allogeneic
Surgery-Induced Diabetes (Iatrogenic)
Chronic pancreatitis	ITA	Autologous/Allogeneic
Trauma	ITA	Autologous/Allogeneic
Multi-visceral transplantation	Different combinations: Liver-Islet Transplantation, Bowel-Liver-Islet Transplantation, etc.	Allogeneic
Cystic Fibrosis	ITA Lung-Islet Transplantation	Autologous/Allogeneic Allogeneic
Benign enucleable tumors	ITA	Autologous
Borderline/malignant pancreatic neoplasms	ITA	Autologous/Allogeneic
Grade C pancreatic fistula requiring completion pancreatectomy	ITA	Autologous/Allogeneic

ITA- Islet Transplant Alone; SIK- Simultaneous Islet-Kidney; IAK- Islet After Kidney

The current main indication for an allogeneic islet transplant is Type 1 Diabetes, which is characterized by the selective destruction of islet beta cells due to an autoimmune process. Ongoing clinical trials of allogeneic islet transplantation are recruiting subjects with unstable Type 1 Diabetes 18-65 years of age, either sex, with frequent metabolic instability requiring medical treatment (hypo-, hyper-glycemia, ketoacidosis) despite intensive insulin therapy; hypoglycemia unawareness (<54mg/dL); severe metabolic lability (mean amplitude of glycemic excursion >11,1 mmol/L or 200 mg/dl). The inadequate efficacy of medical therapy to attain the desirable metabolic control in this specific patient population with unstable diabetes justifies the use of transplantation of pancreatic islets (either isolated cellular graft or vascularized whole pancreas) (48). The main objective of the transplant is to correct the high susceptibility to severe hypoglycemia and glycemic imbalance that are associated with high mortality (8% in nonuremic subjects in the waiting list for 4 years to receive pancreas transplantation). Further indications for an islet transplant are presence of progressive complications of diabetes and psychological problems with insulin therapy that may compromise adherence to the therapeutic regimen. Islet transplant is indicated also for cases of subcutaneous insulin resistance requiring intraperitoneal or intravenous infusions, which are associated with substantial management hurdles and morbidity.

Table 2. Inclusion and Exclusion Criteria for Allogeneic Islet Transplantation in T1DM*

Inclusion Criteria:

· Mentally stable and able to comply with study procedures

· Clinical history compatible with type 1 diabetes with onset of disease at <40 years of age, insulin dependence for at least 5 years at study entry, and a sum of age and insulin dependent diabetes duration of at least 28

· Absent stimulated C-peptide (<0.3 ng/ml) 60 and 90 minutes post-mixed-meal tolerance test

· Involvement of intensive diabetes management, defined as:

o Self-monitoring of glucose values no less than a mean of three times each day averaged over each week

o Administration of three or more insulin injections each day or insulin pump therapy

o Under the direction of an endocrinologist, diabetologist, or diabetes specialist with at least three clinical evaluations during the past 12 months prior to study enrollment

· At least one episode of severe hypoglycemia in the past 12 months, defined as an event with one of the following symptoms: memory loss; confusion; uncontrollable behavior; irrational behavior; unusual difficulty in awakening; suspected seizure; seizure; loss of consciousness; or visual symptoms, compatible with hypoglycemia in which the individual required assistance of another subject was unable to treat him/herself person and which was associated with either a blood glucose level <54 mg/dl or prompt recovery after oral carbohydrate, intravenous glucose, or glucagon administration in the 12 months prior to study enrollment

· Reduced awareness of hypoglycemia

Exclusion Criteria:

· Body mass index (BMI) >30 kg/m² or weight ≤50 kg

· Insulin requirement of >1.0 IU/kg/day or <15 U/day

· HbA1c >10%

· Untreated proliferative diabetic retinopathy

· Systolic blood pressure >160 mmHg or diastolic blood pressure >100 mmHg

· Measured glomerular filtration rate using iohexol of <80 ml/min/1.73mm².

· Presence or history of macroalbuminuria (>300 mg/g creatinine)

· Presence or history of panel-reactive anti-HLA antibody levels greater than background by flow cytometry.

· Pregnant, breastfeeding, or unwilling to use effective contraception throughout the study and 4 months after study completion

· Presence or history of active infection, including hepatitis B, hepatitis C, HIV, or tuberculosis.

· Negative for Epstein-Barr virus by IgG determination

· Invasive aspergillus, histoplasmosis, or coccidioidomycosis infection in the past year

· History of malignancy except for completely resected squamous or basal cell carcinoma of the skin

· Known active alcohol or substance abuse

· Baseline Hgb below the lower limits of normal, lymphopenia, neutropenia, or thrombocytopenia

· History of Factor V deficiency

· Any coagulopathy or medical condition requiring long-term anticoagulant therapy after transplantation or individuals with an INR greater than 1.5

· Severe coexisting cardiac disease, characterized by any one of the following conditions:

o Heart attack within the last 6 months

o Evidence of ischemia on functional heart exam within the year prior to study entry

o Left ventricular ejection fraction <30%

· Persistent elevation of liver function tests at the time of study entry

· Symptomatic cholecystolithiasis

· Acute or chronic pancreatitis

· Symptomatic peptic ulcer disease

· Severe unremitting diarrhea, vomiting, or other gastrointestinal disorders that could interfere with the ability to absorb oral medications

· Hyperlipidemia despite medical therapy, defined as fasting LDL cholesterol >130 mg/dl (treated or untreated) and/or fasting triglycerides >200 mg/dl

· Currently receiving treatment for a medical condition that requires chronic use of systemic steroids except for the use of 5 mg or less of prednisone daily, or an equivalent dose of hydrocortisone, for physiological replacement only

· Treatment with any antidiabetic medication other than insulin within the past 4 weeks

· Use of any study medications within the past 4 weeks

· Received a live attenuated vaccine(s) within the past 2 months

· Any medical condition that, in the opinion of the investigator, might interfere with safe participation in the trial

o Treatment with any immunosuppressive regimen at the time of enrollment.

o A previous islet transplant.

· A previous pancreas transplant, unless the graft failed within the first week due to thrombosis, followed by pancreatectomy and the transplant occurred more than 6 months prior to enrollment.

*Modified from the information relative to active trials from the Clinical Islet Transplant Consortium (www.citisletstudy.org/) as listed at http://clinicaltrials.gov/ct2/show/NCT00434811.

MULTIDISCIPLINARY TEAM

Islet Transplant Programs require the integration of multidisciplinary expertise. The endocrinologist expert in diabetes diagnosis and management is essential member of the team, and can identifying subjects who may benefit of beta-cell replacement therapy, and help with the evaluation of metabolic control during all phases of the follow-up. The psychologist is involved in the evaluation of islet transplant candidates to assess their motivation, mental fit to enroll in the trial, and ability to adhere to the therapy. Psychometric and psychological evaluations are performed during the follow-up period after transplantation. Transplant surgeons provide the expertise in organ procurement, with transplant procedures, overall management of patients and immunosuppression. A dedicated Cell Transplant Center with specialized experts in pancreatic cell isolation, purification, culture, potency assessment and quality assurance warrant that islet cell products are manufactured for clinical transplantation following cGMP standards and FDA regulations. The interventional radiologist performs the noninvasive cannulation of the portal vein and participates to the post-transplant monitoring of the liver using noninvasive imagine techniques. The organ procurement organizations and organ distribution networks (UNOS in the U.S.) contribute to the identification and allocation of donor organs matching the recipient’s characteristics. The ophthalmologist and nephrologist are involved to monitor and treat progressive diabetic complications (i.e., retinopathy and renal function, respectively).

ISLET ISOLATION AND TRANSPLANTATION

Islets are highly vascularized cell clusters ranging <50um to ~800um of diameter that constitute the endocrine component of the pancreas. It has been estimated that a healthy pancreas may contain approximately 10⁶ islets scattered throughout the gland, and accounting for only ~1% of total pancreatic tissue. Each cluster comprises several thousands of endocrine cell subsets that are closely in touch with capillaries and with each other. Complex cell-cell interactions between different cell subsets, innervation, incretins and metabolites (sugar and amino acids, amongst other) in the blood and interstitial space all contribute to the proper control of glucose homeostasis (49). Preservation of the integrity of islet cell cluster is a prerequisite for their optimal function. The procedure currently used to extract islets from human pancreas is the so called automated method for isolation of the islets of Langerhans, established in 1987 by Ricordi and colleagues (50). Before the beginning of the isolation procedure, the spleen and the duodenum are removed from the pancreas and an accurate dissection and removal of the peripancretic fat, lymph nodes and vessels is performed. Then, the pancreas is divided at the neck and two 16-20 gauge angiocatheters are inserted into the main pancreatic ducts. The organ is then perfused with cooled perfusion solution containing collagenase and serine – protease inhibitor – dissolved in buffer at a pressure of 140-180 mmHg. After 10 minutes of cold perfusion, the distended pancreas is further cut into smaller sections, and placed into the Ricordi chamber. This chamber is composed of a superior and an inferior part, separated by a filter that has pores of about 700μm. Seven to nine stainless steel balls and the fragments of the pancreas are placed into the inferior part of the chamber, which is then filled with the digestion solution and closed together with the superior part of the chamber. A peristaltic pump connected to the system is activated creating a flow of 40 ml/min. The digestion runs in a closed circuit where warm Hank’s solution is pumped in the inferior chamber and the tissue released in the solution passes in the superior chamber through the filter. The collagenase is re-circulated at a temperature not exceeding 37°C and the chamber is agitated. When most of the islets are free of the surrounding acinar tissue, and intact islets are observed, the heating circuit is bypassed. The temperature is progressively decreased to 10°C and the collagenase diluted with cold RPMI. The free islets are then collected in containers, washed several times, re-suspended in cold organ preservation solution and purified with a continuous ficoll gradient using a Cobe 2991 cell separator. At the end of the procedure samples of the islet preparation are collected and evaluated through staining with dithizone (DTZ) which marks zinc in the insulin granules, resulting in a characteristic red stain. Adding few drops of DTZ solution to a sample allows easy evaluation of the morphology and number of isolated islets through computerized digital analysis. The islet manufacturing processes must be controlled by different assays and the islet batch product validated and characterized. Then safety testing is carried out for sterility and pyrogenicity, identity (insulin content), cell number (amount of tissue, counting of islets), purity (percentage of ductal, acinar, beta, and other cells), viability (islet nucleotide content), potency (insulin secretory response) and finally stability (storage in culture). Specific features of the final islet preparation are a required for islet preparations used in islet transplantation, in particular purity (> 20% of the preparation being islets), adequate number of islets (>5,000 islet equivalent recipient body weight for the first infusion, >3,000 for further infusions) and total tissue volume (< 5 ml). The infusion of the islets can be performed a few hours after the end of the isolation process or up to 72 hours thereafter. The implantation site is usually the hepatic parenchyma through the portal system of the recipient. Recently other implantation sites have been proposed (51) in the clinical setting, like the bone marrow (52,53), the subcutaneous site(54), the gastric submucosa (55), the omentum(56,57) or striated muscle (58,59), which in the future, may prove to be valid alternative sites for islet transplantation. The adequate amount of islets obtained is calculated with respect to the body weight of the recipient and re-suspended immediately before intrahepatic transplantation in 40-60 mL of a solution suitable for injection (Ringer Lactate, 1% Human Albumin and 2000 IE of heparin). Percutaneous trans hepatic catheterization is the most common access route, as well as a mini-laparotomy and cannulation of an omental or mesenteric vein, or recanalization of the umbilical vein. Access to the portal vein is usually provided by interventional radiologists. If the portal pressure is documented to be below 20 mmHg, the islet infusion bag is connected with the portal vein catheter and infused over a period of 15 to 60 minutes. Islet infusion is halted if the portal pressure exceeds 22 mmHg. After completion of the islet infusion, the catheter is withdrawn; coils and gelatin-sponges are deployed in the puncture tract to prevent bleeding. A schematic animation of the islet isolation and transplant procedures is available online [http://www.youtube.com/watch?v=aMNKu-ZVUls].

THE CONSORTIUM CONCEPT

A major development in the field of islet transplantation is the combination of individual centers into larger groups such as the GRAGIL network in France and Switzerland, the Nordic Network for Clinical Islet Transplantation (NNCIT) in the Scandinavian countries and the Clinical Islet Transplant Consortium (CITC) internationally but concentrated in North America. The need for dedicated infrastructures and personnel specialized in islet cell processing, quality assessment and cGMP standards impose an enormous financial burden on any Clinical Islet Transplant Program. Acquiring and maintaining the specialized expertise in islet cell processing requires a steep learning curve and continuous refinements and training that add to the costly procedure. Recent data have shown that the experience of the clinical islet transplant team in cell processing and management of immunosuppression are critically important in determining the success of a clinical trial (60). Based on these premises, the development of regional cell processing centers that are part of consortia that are integrated with distant transplant centers is increasingly being considered as a practical and cost-effective strategy (Figure 1). Initial reports of successful clinical trials carried on in the context of Consortia both in Europe and North America (61-64) support the feasibility of such an approach, which may be of assistance in reducing the operational costs while enhancing the success rate of clinical trials (i.e., better utilization of donor pancreata, more reproducible success in obtaining adequate numbers of functional islets from a donor pancreata, etc.).

Figure 1. Islet Transplant Consortium Models. A. The centralized (or ‘regional’) Cell Processing Facility receives the donor pancreas from a distant Transplant Center and isolates islet cell products that are sent back for implant. B. The centralized Cell Processing facility receives the donor pancreas from one of the Transplant Centers and distributes the isolated islets to any of the Transplant Center in the Consortium according to the best match of the cell product for the transplant candidate on the waiting list for transplant (that is, the islet cell product is not necessary returned to the center recovering the pancreas).

ISLET TRANSPLANT ACTIVITY

The Collaborative Islet Transplant Registry (CITR)

In 2001, the National Institute of Diabetes & Digestive & Kidney Diseases established the Collaborative Islet Transplant Registry (CITR) to compile data from all islet transplant programs in North America from 1999 to the present. The Juvenile Diabetes Research Foundation (JDRF) granted additional funding to include the participation of JDRF-funded European and Australian centers from 2006 through 2015. The cumulated North American, European and Australian data are pooled for analyses included in the annual report. CITR Annual Reports are publicly available as open access and can be downloaded or requested in hard copy at www.citregistry.org. From 1999 through 2020 – the cut-off for the last Eleventh Annual Report – CITR has collected data on the following groups of study subjects:

Allogeneic islet transplantation (typically cadaveric donor), performed as either islet transplant alone (ITA) or islet-after-kidney (IAK). A small number of cases have been performed as islet simultaneous with kidney (SIK) or kidney-after-islet (KAI).
Autologous islet transplantation, performed after total pancreatectomy (N=1,233) are also reported to CITR.

As of December 15, 2020, the CITR Registry included data on 1,399 allogeneic islet transplant recipients (1,108 islet transplant alone, ITA, and 236 islet after kidney, IAK, 49 simultaneous islet kidney, SIK, and 6 kidney after islet, KAI), who received 2,832 infusions from 3,326 donors. From 1999 through 2020, 28 National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored North American and 12 international Eurasian and Australian islet transplant centers (40 total) contributed data to the Collaborative Islet Transplant Registry (CITR). Combining the ITA and IAK recipients, 27.4% received a single islet infusion, 48.1% received two, 20.4% received three, and 4.1% received 4-6 infusions. Of 26 North American sites performing Auto-ITx from 1999 through December 2020, 15 reported data to CITR along with 5 European and Australian islet transplant centers. These sites registered 1233 autoislet transplant recipients. Of these, 1123 recipients were in North America, 98 in Europe, and

12 in Australia. One-hundred eight-five (185) were aged less than 18, and 1,057 were 18 or older at the time of their transplant.

Outside The Collaborative Islet Transplant Registry (CITR)

Although the CITR is an extraordinary source of valuable data, a recent publication indicates that it does not capture a major part of the international islet transplant activities and outcomes (65). In fact, a global online survey was recently administered to 69 islet transplantation programs. After integration of all data obtained, 103 islet transplant centers were identified, of which 94, in 25 countries, had reported allotransplantation activity during the 2000–2020 period: 15 in Asia (16%), 39 in Europe (42%), 34 North America (36%), 3 in Oceania (3%) and 3 in South America (3%) and between January 2000 and December 2020, 4,321 islet allotransplants in 2,149 patients were reported worldwide. Most islet transplants were performed in Europe (2,608, 59.7%), followed by North America (1,475, 33.8%), Asia (135, 3.1%), Oceania (119, 2.7%) and South America (28, 0.6%). Actually the ANZIPTR (Australia and New Zealand Islet and Pancreas Transplant Registry) and NHS-BT (UK National Health Service-Blood and Transplant) registry are publicly available registries containing a wealth of data on islet and pancreas transplantation in Australia/New Zealand and UK, respectively, including outcomes (66) (67). The European Pancreas and Islet Transplant Registry (EPITR) is a current effort from ESOT/EPITA aiming at covering these needs for Europe (https://esot.org/epita/epita-epitr/).

Clinical Management of Islet Transplant Recipients

The clinical management of islet transplant recipients requires the concerted effort of endocrinologist and transplant teams.

Immunosuppression

Preexisting and transplant-induced auto- and allo-specific cellular immune responses play a crucial role in the loss of islets and islet function infused in the liver (68-70) along with non-specific immune responses predominantly mediated by innate inflammatory processes related to mechanics and site (71-74). Islet graft rejection occurs without clinical symptoms. Neither guidelines nor formal consensus on the “best” or “standard” immunosuppressive strategy for human islet transplantation are currently available. Multiple induction and maintenance agents are administered peri- and post- every infusion in the same recipient. According to CITR data (75), a substantial shift in immunosuppression strategies has been documented during the last years.

Induction with interleukin-2 receptor antagonist (e.g., daclizumab) only, which comprised about 53% of all initial infusions in 1999-2002, was replaced or supplemented with regimens that included T-cell depletion with/without TNF antagonists in about 67% of the new infusions performed since 2015 (11,76-83). In 1999-2002, maintenance immunosuppression was predominantly (64%) calcineurin (CNI) +mTOR inhibitors (60). It was increasingly replaced or supplemented throughout the eras by a CNI and IMPDH-inhibitor combination (77,84-86).; in the most recent era, CNI+mTOR inhibitors were used in 15% of new infusions while CNI+IMPDH inhibitors were used in about 62%. Moreover, the use of alemtuzumab-induction therapy was recently reported and associated with encouraging longer-term function (87,88). New biologic agents with potentially lower islet cell and organ toxicity profiles are currently being evaluated in ongoing clinical trials. Amongst these are agents that target co-stimulation pathways in immune cells and/or adhesion molecules (CTLA4-Ig, LFA-1 PD-1/PD-L1 CD40 ) (89-95) or chemokine receptors (CXCR1/2) (71,96). Finally, calcineurin inhibitor-free immunosuppressive regimen was reported (97).

Antibiotic and Antiviral Prophylaxis

Subjects receiving immunosuppression therapy are more susceptible to opportunistic infections, as well as reactivation or de novo occurrence of viral infections. Antibiotic prophylaxis for Pneumocystis carinii consists in trimethoprim and sulfamethoxazole three times a week. Antiviral prophylaxis is aimed are reducing the risk, or treating the occurrence, of cytomegalovirus infections (which have been recognized increasing the risk of graft loss in solid organ transplantation) and of reactivation of Epstein - Barr virus infection (which has been associated with the dreadful post-transplant lymphoproliferative disease, PTLD). Current protocols utilize antiviral therapy with vanglancyclovir daily for the first trimester post-transplant. Monitoring of viremia in peripheral blood samples by PCR is becoming a routine during the follow-up as it allowed for the early detection of reactivation or de novo infections that may be treatable without compromising graft outcome (98,99).

Thromboembolism Prophylaxis

It has been recognized that isolated islets produce tissue factor and other pro-inflammatory molecules that may trigger an instant blood-mediated inflammatory reaction upon infusion into the blood stream. This, in turn, may enhance the generation of noxious stimuli after embolization in hepatic sinusoids of the liver, significantly reducing the mass of functional islets engrafting. An aggressive heparin treatment is generally implemented in the early period after transplant. Heparin is added to the transplant medium used during the islet infusion, while low molecular weight heparin injections are administered in the post-transplant period. This is aimed at enhancing islet engraftment in the hepatic portal system while reducing the risk of portal thrombosis.

Peri-transplant Insulin Management

Islet engraftment may take up a few weeks to allow for neovascularization of the clusters in the transplant microenvironment. The monitoring of glycemic control in the immediate post-transplant period should be intense to attain tight glycemic values in order to avoid excessive workload for the newly transplanted islets as well as to prevent hypoglycemic episodes. This is generally done by providing basal exogenous insulin that is then progressively reduced and withdrawn according to the glycemic values measured.

POST-TRANSPLANT CLINICAL MONITORING

Monitoring of cell blood counts (erythrocytes, white blood cells and differential), hemoglobin, platelets and coagulation markers is routinely performed in the post-transplant period. These tests allow assessing the myelosuppressive effects of anti-rejection drugs. In the case of anemia with clinical relevance, iron supplementation may be indicated, while for more severe cases erythropoietin treatment is implemented. In the case of severe neutropenia, marrow stimulation with granulocyte-colony stimulating factor (G-CSF) is promptly implemented.

Renal function is monitored periodically in the follow-up of islet transplant recipients to assess the impact of restoring beta-cell function on the progression of diabetic nephropathy, and also to identify and timely correct potential nephrotoxicity of anti-rejection drugs (i.e., CNI and mTOR inhibitors). Standard serological tests (serum creatinine, azotemia), urine tests (spot and 24-hr collections) are frequently performed during the follow-up and glomerular filtration rates (GFR) estimated using different algorithms (i.e., MDRD). The nephroprotective effect of ACE inhibitors and of antagonists of angiotensin-receptor (ARB) in subjects with diabetes has been recognized, and their use is particularly indicated in transplant recipients treated with immunosuppressive drugs known for their negative effects on renal function (100-104). Elevations of blood pressure from the range 130/80 mmHg are promptly treated pharmacologically.

Monitoring of lipid levels and prompt treatment of dyslipidemia are important in transplanted patients. Some of the anti-rejection drugs (i.e., mTOR inhibitors) are prone to induce dyslipidemia, which in turn may have toxic effects on beta-cells or contribute to creating an unfavorable environment (i.e., steatosis) in the liver (105). Prophylactic use of statins targeting LDL cholesterol levels <100mg/dL can be contemplated for islet transplant recipients.

Liver function is monitored in the post-transplant period. It is common to observe a transient and self-limited elevation of liver enzymes (transaminitis) because of the embolization of islets into the liver sinusoids (106,107). This is often associated with hyper-echoic pattern of the liver parenchyma at ultrasound evaluation in the early days post-transplant. This phenomenon resolves spontaneously without the need for medical treatment. Ultrasound evaluation of the liver and abdominal cavity in the days post-transplant also allows identifying timely possible procedural complication of the transplant, such as portal thrombosis, peritoneal hemorrhage and alterations of echogenicity of hepatic parenchyma (108).

The immune monitoring after islet transplantation does not differ much from that of any other organ transplant (109). Basal and serial evaluation of Panel Reactive Antibodies (PRA) is performed to determine possible allosensitization against Human Leukocyte Antigens (HLA) class I and II of the Histocompatibility complex of transplanted tissue. Generally, maintenance of an adequate immunosuppressive regimen can prevent the development of alloantibodies, thereby preventing their deleterious effects on graft survival and function (i.e., chronic rejection leading to graft loss) (110-112). Nonetheless, development of alloreactivity against donor or non-specific antigens may develop whenever reduction (i.e., during infections, toxicity and drug conversion, amongst other causes) or suspension (i.e., after complete graft loss) of immunosuppression is needed (112,113). The autoimmune process underlying Type 1 Diabetes is associated with the appearance of antibodies against self-antigens (autoantibodies; i.e., towards GAD, IA-1 and insulin). Serial titration of autoantibody levels during the follow-up period may enable detecting a reactivation of the autoimmune process, measured as conversion to positive values in previously negative subjects, or increase of antibody titers. These have been associated with a lower rate of insulin independence and shorter duration of graft function after islet transplantation (60,70). New assays for additional autoantibodies (i.e., ZnT8) and for autoreactive T cells are under evaluation to help enhancing the sensitivity of immune monitoring for early detection of recurrence of autoimmunity, which may enable implementation of timely immune interventions to rescue the transplanted cells (114-116).

MONITORING ISLET GRAFT FUNCTION

Several metabolic parameters allow monitoring the function of transplanted islets (Table 3). Since only subjects with Type 1 Diabetes who have undetectable stimulated c-peptide (<0.3 ng/dl) before transplant are recruited for an islet transplant, monitoring of basal and stimulated c-peptide offers an excellent biomarker of graft function, even when exogenous insulin is required. There is no consensus on which approach is most suited to accurately assess functional islet mass. Algorithms and indices that combine multiple parameters have been developed and proposed to help simplifying and obtaining objective functional assessment of islet transplant recipients (117-126). The main goal is to identify early changes that indicate propensity to graft dysfunction (i.e., functional impairment during an infection, drug-induced toxicity). Stimulation tests are performed before (at enrollment) and during the follow-up after transplant to evaluate the functional potency of the transplanted islets in response to different secretagogues (i.e., glucose, arginine, or mixed meal test). Insulin therapy is generally implemented when random glycemic sampling demonstrates on three subsequent occasions within the same week fasting values >140 mg/dl (7.8 mmol/L) and postprandial values >180 mg/dl (10.0 mmol/L), or after recording two consecutive A1c values >6.5%.

Table 3. Monitoring of Islet Graft Function
Standard	Stimulation	Indices
Glycosylated Hb (A1c) Fasting glycemia Postprandial glycemia MAGE* CGMS* Basal C-peptide Daily insulin requirement	Mixed Meal Intravenous glucose Intravenous arginine	Hypo score Liability index Βeta score Beta 2 score Basal C-peptide/Glucose ratio HOMA-B* HOMA-IR* TEF*
Abbreviations. CGMS: Continuous Glucose Monitoring System. MAGE: Mean Amplitude of Glucose Excursions*. HOMA-B: Homeostasis Model Assessment – functional beta cell mass. HOMA-IR: Homeostasis Model Assessment – Insulin-Resistance. TEF: Transplant Estimated Function

The Igls Score

The lack of standardized definition of graft functional and clinical outcomes remains a source of concern in β-cell replacement influencing its recognition as a valid clinical option from the endocrinology community. In order to address this issue, the International Pancreas & Islet Transplant Association (IPITA) joined with the European Pancreas & Islet Transplant Association (EPITA) for a two-day workshop on “Defining Outcomes for β-Cell Replacement Therapy in the Treatment of Diabetes” in January 2017 in Igls, Austria. The main objective was to develop consensus on the definition of function and failure of current and future forms of β-cell replacement therapies. As result of the workshop, an IPITA/EPITA Statement was recently published (127,128). This Statement introduces some relevant innovations in the field including a new classification for the definition of clinically successful outcome. The functional status and clinical success of a β-cell graft should be defined separately using the same components of assessment: the HbA_1c, severe hypoglycemic events, insulin requirements, and C-peptide. Concordantly, a four-tiered system was proposed to classify the functional outcomes of β-cell replacement:

optimal β-cell graft function: HbA_1c ≤6.5%, the absence of any severe hypoglycemia, the absence of any requirement for exogenous insulin or other anti-diabetic drugs, and documentation of an increase over pre-transplant measurement of C-peptide
good β-cell graft function: defined by: HbA_1c <7.0%, the absence of any severe hypoglycemia, a reduction by more than 50% from baseline in insulin requirements or the use of non-insulin anti-diabetic drugs, and documentation of an increase over pre-transplant measurement of C-peptide.
marginal β-cell graft function: no modification of HbA_1c, the reduction of severe hypoglycemia, a reduction by less than 50% from baseline in insulin requirements, and documentation of an increase over pre-transplant measurement of C-peptide.
failure β-cell graft function: absence of any evidence for a clinical impact (no modification of HbA_1c,incidence of severe hypoglycemia and insulin requirement) and clinically insignificant levels of C-peptide.

Clinically successful outcomes includes both optimal and good functional outcomes, implying that the use of exogenous insulin or other anti-diabetic drugs is not synonymous with graft loss or failure. Neither a marginal β-cell graft nor a failed β-cell graft is considered a clinically successful. However, if documented impairment in hypoglycemia awareness, frequent occurrence or exposure to severe hypoglycemia, or marked glycemic variability/lability is convincingly improved, then it may be appropriate to consider that the β-cell graft is clinically impactful also in marginal function and the benefit of maintaining β-cell graft function may outweigh risks of maintaining immunosuppression. This implies that hypoglycemia awareness, serious hypoglycemia, and glycemic variability/lability must be evaluated at baseline for monitoring whether a marginally functioning graft is continuing to provide any clinical impact.

IPITA / EPITA Statement has the merit of having introduced a defined concept of clinical success based on easily measurable parameters over time and with a wide consensus of international experts Implementation of this new β-cell replacement outcome definition and its use in publication will be critical to improve the performance and to reliably compare the different β-cell replacement strategies (129).

In July 2019, a symposium at the 17th IPITA World Congress was held to examine the Igls criteria after 2 years in clinical practice, including validation against continuous glucose monitoring (CGM)-derived glucose targets, and to propose future refinements that would allow for comparison of outcomes with artificial pancreas system approaches. A new Igls 2.0 form composite criteria was suggested (130), in which clinical outcome based on glucose regulation is separated from β-cell graft function, with the latter considered only for further qualification of β-cell replacement modalities (Table 4-5).

Table 4. Proposed Igls Criteria 2.0
Treatment outcome	Glycemic control		Hypoglycemia		Treatment success
	HbA_1c, % (mmol/mol)^a	CGM, % time-in-range	Severe hypoglycemia, events per y	CGM, % time < 54 mg/dl (3.0 mmol/L)
Optimal	≤6.5 (48)	≥80	None	0	Yes
Good	<7.0 (53)	≥70	None	<1	Yes
Marginal	≤Baseline	>Baseline	<Baseline^b	<Baseline	No^c
Failure	~Baseline	~Baseline	~Baseline^d	~Baseline	No

Baseline, pretransplant assessment (not applicable to total pancreatectomy with islet autotransplantation patients).

Abbreviations: CGM, continuous glucose monitoring; HbA1c, glycated hemoglobin.

a Mean glucose should be used to provide an estimate of the HbA1c, termed the glucose management indicator, in the setting of disordered red blood cell life span.

b Should severe hypoglycemia occur following treatment, then continued benefit may require assessment of hypoglycemia awareness, exposure to serious hypoglycemia (<54 mg/dL [3.0 mmol/L]), and/or glycemic variability/lability with demonstration of improvement from baseline.

c Clinically, benefits of maintaining and monitoring β-cell graft function may outweigh risks of maintaining immunosuppression.

d If severe hypoglycemia was not present before β-cell replacement therapy, then a return to baseline measures of glycemic control used as the indication for treatment (6, 7) may be consistent with β-cell graft failure.

Table 5. Proposed Igls Criteria 2.0
β-cell graft function^e	C-peptide, ng/mL (nmol/L) ^f	Insulin use or noninsulin antihyperglycemic therapy
Optimal	Any	None
Good	>0.5 (0.17) stimulated ≥0.2 (0.07) fasting	Any
Marginal	0.3-0.5 (0.10-0.17) stimulated 0.1-<0.2 (0.04-<0.07) fasting	Any
Failure	<0.3 (0.10) stimulated <0.1 (0.04) fasting	Any

^eCategorization of β-cell graft function must first meet treatment outcome based on measures of glucose regulation.

^fMay not be reliable in uremic patients and/or in those patients with evidence of C-peptide production before β-cell replacement therapy.

IMPACT OF ISLET TRANSPLANTATION ON METABOLIC CONTROL (TABLE 6)

Four successful large-scale Phase 3 clinical trials in islet transplantation have been published recently: CIT-07 (multicenter, single-arm)(131), CIT06 (pivotal trial) (132), TRIMECO (multicenter, open-label, randomized) (133) and REP0211 (multicenter, Double blind, randomized) (134). All these studies demonstrate that human islets, when transplanted in patients with T1D with impaired awareness of hypoglycemia and severe hypoglycemic events, can safely and efficaciously maintain optimal glycemic control (135). The clinical experience confirms that the most remarkable effect of the islet transplant is the abrogation of severe hypoglycemia and the recovery of hypoglycemia awareness, which persists after development of graft dysfunction and even several months after graft failure (and loss of detectable c-peptide) (136,137). Following islet transplantation, the restoration of β cell responses to secretagogue stimulation is observed, with improved insulin secretion (‘first phase’) in response to intravenous glucose, as well as increased c-peptide secretion in response to oral glucose. Normalization of glycemic threshold triggering the release of counter-regulatory hormones can be demonstrated during hypoglycemic clamp studies, even if without reaching normalization of the magnitude of the vegetative response; furthermore, quasi-normal glucagon secretion in response to hypoglycemia can be observed (138-142). In addition to controlling hypoglycemia, insulin independence can be achieved when an adequate islet mass is transplanted (143). After islet transplantation, 5-year insulin independence may be as high as 50%. A quarter of patients may remain insulin independent, with HbA1c concentrations of less than or equal to 6·5%, for at least 10 years, with either islet transplantation alone or islet-after-kidney transplantation (144)(145). Moreover, the glucose control associated with excellent islet graft function closely matches glucose values measured in healthy adults: median glucose 103 mg/dl (95-112), glucose standard deviation around the mean value 14 (11-20), 0% time >180 mg/dl, 0% time <54 mg/dl, HbA1c 5.6 (5-5.8) (146). Additionally, a significant improvement of quality of life after islet transplant has been documented by using standardized psychometric instruments and interviews carried on by psychologists (147-155). Associated with the better glucose control and the evidence of islet function (c-peptide secretion), a positive impact on the microvascular complications of T1D has been described while is less evident on the macrovascular complications (156). More specifically, a stabilization/slower progression of retinopathy (104,157-159) and neuropathy (158,160-162) and an improvement of micro- and macroangiopathy (79,101,102,154,157,163-168) have been described. Some studies reported also a reduction of atherothrombotic profile paralleled by reduced incidence of cardiovascular accidents, an amelioration of cardiovascular and endothelial function, improved longevity of renal transplant (165) and a higher survival rates after islet transplantation in IAK recipients (162,165,169-173).

By combining donor selection criteria with improved isolation techniques and adequate immunomodulation of the recipient, insulin independence after single donor islet preparation is becoming more reproducibly possible to achieve. Islet preparations obtained from more than one donor pancreas can be transplanted at once after pooling them, or sequentially based on the metabolic needs of each subject. Data from the Clinical Islet Transplant Registry and independent trial reports have shown that insulin independence at one year from completion of the transplant is up to 70% with virtually 100% of the subjects maintaining graft function (c-peptide) if adequately immunosuppressed (75,82). A progressive loss of insulin independence with approximately 90% of subjects requiring reintroduction of exogenous insulin (most of them with detectable c-peptide) has been reported in recent clinical trials based on the ‘Edmonton protocol’ (induction with anti-IL2R antibody; maintenance with sirolimus and tacrolimus) and some variants of it (60,77,84,86,174). More recent trials using more potent lymphodepletion (i.e., thymoglobulin, anti-CD3 or anti-CD52 antibodies) and/or biologics (anti-IL2R, anti-TNF, anti-LFA-1 antibody or CTLA4Ig) have shown great promise with approximately 50% of insulin independence at 5 years after islet transplantation (86,175-179), which is comparable to some of the data in whole pancreas transplantation in subjects with Type 1 Diabetes (80,83,86,91,92). In light of the results of the last decade of clinical islet transplant trials, achievement of insulin independence, although desirable, no longer should be considered the main goal of islet transplantation. The sizable improvement of metabolic control in the absence of severe hypoglycemic events, the amelioration of diabetes complications and the achievement of sustained better quality of life, which are quite cumbersome to reproduce by the means of medical treatment, justify the risks associated with the islet transplant procedure and immunosuppression in this high-risk population of subjects with unstable diabetes.

Regarding auto transplantation the largest published series are from the University of Minnesota (16-19), University of Cincinnati (20,21), and Leicester (22-25,180). Overall, one-third of patients in the Minnesota series achieves insulin independence, and the majority have islet graft function, as documented by C-peptide positivity (16,22). Cincinnati, Leicester, and other centers have published similar results, with 22-40% of the patients being insulin independent after islet transplant (21,181,182). A significant association between insulin independence and the IEQ/kg transplanted (i.e., islet mass standardized by patient’s weight) was described. Bellin et al. (19) and White et al. (24) reported that insulin independence is related to the number of transplanted islet cells (>2,000 IEQ/kg and >3,000 IEQ/kg, respectively). Similarly, Sutherland et al. (183) reported that insulin independence at 1 year was observed in 63 % of the patients who received greater than 5,000 IE/kg. Moreover, pancreatectomy recipients benefit from an islet autograft ways apart from insulin independence. In fact, the major goal of IAT in these patients is a good glycemic control without brittle diabetes. Ninety percent of patients in the Minnesota series and 100% of those in the Leicester series were C-peptide positive after the procedure (16,22). The majority of patients receiving an islet auto transplant maintained good glycemic control, with 82% of all recipients having average HbA1c levels <7.0% (16).

IMPACT OF ISLET TRANSPLANTATION ON DIABETES COMPLICATIONS

Encouraging results have been reported in recent years on the multiple beneficial effects of islet transplantation on the progression of diabetes complications [reviewed in (184)]. Although based on nonrandomized pilot studies, which should be cautiously evaluated, they provide the proof of concept of the importance of restoring beta-cell function in patients with diabetes. In particular, improvement of micro- and macro-angiopathy (main causes of diabetic nephropathy) (79,101,102,154,157,163-168) and stabilization/reduced progression of retinopathy (104,157-159) and neuropathy (158,160-162) have been described. Amelioration of cardiovascular and endothelial function, reduction of atherothrombotic profile paralleled by reduced incidence of cardiovascular accidents and higher survival rates were reported In IAK recipients (169-172) (162,165,169,171,173). Furthermore, significantly improved longevity of a renal transplant was observed after islet transplantation (165). It is likely that these benefits are the consequence of improve metabolic control conferred by the islet transplant. In addition, it has been proposed a contribution of restored c-peptide secretion and its effects on multiple targets (185).

Table 6. Benefits of Islet Transplantation

Metabolic control

- Reduction of exogenous insulin requirements or insulin independence

- Reduction of MAGE

- Reduction or normalization of A1c

- Absence of severe hypoglycemia

Quality of Life

- Reduced fear of hypoglycemia

- Improvement of Diabetes Quality of Life

Diabetes complications

- Improvement of micro- and micro-angiopathy

- Improvement of cardiovascular and endothelial function

- Reduced incidence of acute cardiovascular events

- Reduced nephropathy progression

- Stabilization/slower neuropathy progression

- Stabilization/slower retinopathy progression

COMMON ADVERSE EVENTS AND THEIR MANAGEMENT (TABLE 7)

The procedure of islet transplantation has proven to be very safe, especially when compared with whole pancreas transplant (177,186,187). For allogenic islet transplantation bleeding, either intraperitoneal or liver subcapsular, is the most common procedure-related complication, occurring with an incidence as high as 13% (188). The use of fibrin tissue sealant and embolization coils in the hepatic catheter tract seems to effectively minimize the bleeding risk (188,189). Partial portal vein thrombosis complicates fewer than 5% of islet infusion procedures (174), and complete portal venous thrombosis is rare. The use of purer islet preparations, greater expertise in portal vein catheterization and new radiological devices (catheters medicated with anticoagulation) will continue reducing the risk of portal vein thrombosis, although the risk is unlikely be completely eliminated. Other complications of islet cell transplantation include transient liver enzyme elevation (50% incidence) (106), abdominal pain (50% incidence), focal hepatic steatosis (20% incidence) (190,191), and severe hypoglycemia (< 3% incidence). Another complication related to the intrahepatic islet transplantation procedure is portal hypertension that can occur acutely during the islet infusion, especially in the case of infusions other than the first one (192). Finally, severe hypoglycemia is a risk associated with the infusion of islets. Iatrogenic hypoglycemia in the immediate post-transplant period is a rare event. Frequent blood glucose monitoring immediately following islet transplantation is recommended to avoid severe unrecognized hypoglycemia in the early post-transplant period. The risk of transmission of CMV disease from donor to recipient has been surprisingly low in recipients of islet allografts, particularly in the most recent period with routine use of purified islet preparations (140-144). As with any allogeneic transplant, islet transplant recipients may become sensitized to islet donor histocompatibility antigens (HLA), leading to the development of panel reactive alloantibodies (PRA).Data on the development of cytotoxic antibodies against donor HLA in islet allotransplant recipients with failing grafts have been reported from several islet transplant centers (148-152). A potential consequence of high PRA levels in recipients of a failed islet transplants is that if these individuals develop diabetic nephropathy in the future, a high PRA may increase their time on a transplant list for a suitable kidney graft.

The need to implement anti-rejection therapy exposes transplant recipients to an increased risk of untoward side effects expected in any immunosuppressed subjects (Table 6) (107). Opportunistic infections of urinary tract, upper respiratory tract and skin are frequent, along with myelosuppressive and gastrointestinal effects of the immunosuppressive drugs. In the majority of the cases, these effects are not severe and resolve without sequel with medical treatment. Elevation of viremic titers for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) in the presence of overt clinical symptoms (i.e., de novo infection or reactivation in seropositve subjects) imposes the implementation of anti-viral therapy and reduction of immunosuppressive drug dose (98). Timely intervention may result in faster resolution of the symptoms without compromising graft survival. Direct organ toxicity of immunosuppressive drugs has been recognized. Symptoms associated with neuro- and/or nephro-toxicity are relatively frequent in subjects receiving chronic immunosuppressive agents currently in use in the clinical arena. In these cases, modification of the anti-rejection regimen is indicated, with dose reduction or conversion to a different combination of drugs. In the majority of cases, these changes resolve the symptoms without compromising graft survival (193,194). Nephrotoxicity from sirolimus and/or tacrolimus has been described in patients with T1D undergoing islet transplantation, particularly when kidney function is already impaired because of pre-existing diabetic nephropathy (195,196).

As for the CITR 11th Allograft Data Report Scientific Summary, the decline in eGFR (CKD-Epi) after islet transplantation is both statistically significant and clinically important. IAK had much lower pre-transplant levels than ITA, which then declined at a slower rate. Initial levels were also lower in recipients age 35 and older and declined at a slower rate compared to younger recipients. Levels were generally lower among recipients managed with CNI+IMPDH compared to other maintenance immunosuppression regimens. Compared with an age-unadjusted cohort of 1,141 participants with T1D followed by the Diabetes Control and Complications Trial and then by the Epidemiology of Diabetes Interventions and Complications (EDIC) (The DCCT/EDIC Research Group, 2011) who started with mean eGFR levels of 126 ml/min/1.73m3, CITR allograft recipients had much lower mean eGFR (91±1SE for ITA and 62±2 for IAK) at their first transplant. CITR ITA recipients exhibited a decline in eGFR of 12 ml/min/1.73m3 and IAK experienced a mean decline of 2 ml/min/1.73m3 in 5 years from last infusion, compared to a mean decline of about 9 ml/min/1.73m3 over the first 5 years in the DCCT.

As of 2021, by decision of the Executive Committee, only serious adverse events (SAEs) are reportable to CITR. About 11% ITA and 14% of IAK allo-islet recipients experienced a serious adverse event in the first 30 days following transplantation. There was a sharp decline in the number of patients who experienced SAEs post-2010, with 15% or more of patients experiencing SAEs in early eras compared to ~5% in 2011-2014 and 2015-2018. In the first year after islet transplantation, which includes a majority of the reinfusions that were performed, about one-fourth of participants have experienced an SAE. SAE within 1-year was slightly more common in IAK (31%) than ITA (23%) and there was a significant decline post-2010 (>25% pre vs. <15% post). Life-threatening events have occurred in 13.4% of islet-alone, in 16.5% of IAK recipients, and in 20.4% of SIK recipients. Recent eras have seen a substantial decline in the incidence of life-threatening events. The most common life-threatening events reported were abnormal granulocytes (24 events) followed by abnormal liver function (23 events) and hypoglycemia (14 events). About 75% of patients who experienced a life-threatening event recovered fully, 12% recovered with sequelae, 5% did not recover, and 9% died as a result of the event.

A total of 189 instances of neoplasm have been diagnosed in 101 of the 1,399 islet recipients who collectively represent a total of 7,963 person-years of observed follow-up. This equates to about 0.02 neoplasms per person-year. Of the total 189 events, 61% were deemed possibly related to immunosuppression, and 12% definitely related. Of the total events, 69% recovered, 10% did not recover, 5% recovered with sequelae, and 3% resulted in fatality. There were 41 instances in 23 patients of basal carcinoma of the skin and 86 instances in 38 patients of squamous carcinoma of the skin. There were 56 instances in 39 recipients of non-skin cancers. Eleven deaths due to cancer occurred.

There have been 77 or 5.5% deaths; cumulative mortality rates differed significantly by transplant type (p<0.0001) but not by era. SIK transplant recipients were disproportionately represented among fatalities comprising only 3.5% of the allo-islet recipient population, but 15.6% percent of deaths. Of the reported deaths, ten were deemed possibly related or definitely related to islet transplantation or immunosuppression. The most common causes of death were (# cases): cardiovascular (15), neoplasm (11), infection (including pneumonia) (9), hemorrhage (4), and complications of diabetes (3). Twenty-four deaths did not have a cause specified.

An assessment of the surgical complication of islet auto transplantation was recently reported for the entire Minnesota series (n=413) (16). Surgical complications requiring reoperation during the initial admission occurred in 15.9% of the patients. The most common reason for reoperation was bleeding, occurring in 9.5% of the procedures. Anastomotic leaks occurred in 4.2 % of the patients, biliary in 1.4% and enteric in 2.8%. Intra-abdominal infection requiring reoperation occurred in 1.9% of patients, wound infections requiring operative debridement in 2.2%. Gastrointestinal issues, such as bowel obstruction, omental infarction, bowel ischemia, delayed reconstruction because of bowel edema, tube perforation, requiring reoperation in 4.7% of the patients. Two patients (<1%) required reoperation to remove an ischemic or bleeding spleen after spleen sparing pancreatectomy (done in 30% of patients).

Table 7. Most Frequent Complications in Islet Transplant Recipients

Procedure-related

- Hemorrhage

- Portal thrombosis

- Transient transaminitis

Immunosuppression-related

Hematological

- Anemia

- Leucopenia

- Neutropenia

Metabolic

- Dyslipidemia

Gastrointestinal

- Oral ulcers (Sirolimus)

- Diarrhea (Mycophenolic acid)

- CMV colitis

Respiratory tract

- Upper respiratory infections

- Interstitial pneumonitis (Sirolimus)

Neurological

- Neurotoxicity (Tacrolimus)

Genitourinary

- Urinary infections

- Ovarian cysts

- Dysmenorrhea

- Nephropathy

- Proteinuria

Cutaneous

- Infections

- Cancer

CURRENT CHALLENGES

There are many challenges that are currently limiting islet cell transplantation (Table 8) (197-199) While significant progress has been made in the islet transplantation field, several obstacles remain precluding its widespread use. The clinical experience of islet transplantation has been developed almost exclusively using the intra-hepatic infusion through the portal vein (60). It has been suggested that the loss of as many as 50-75% of islets during engraftment is the reason why a very large number of islets are needed to achieve normoglycemia (51,72). Moreover, two additional important limitations are the currently inadequate immunosuppression for preventing islet rejection (70) and the limited oxygen supply to islet in the engraftment site (200,201). Current immunosuppressive regimens are capable of preventing islet failure for months to years, but the agents used in these treatments may increase the risk for specific malignancies and opportunistic infections. In addition, the most commonly used agents (like calcineurin inhibitors and rapamycin) are also known to impair normal islet function and/or insulin action. Furthermore, like all medications, these agents have other associated toxicities, including the harmful effect of certain widely employed immunosuppressive agents on renal function. The second very significant factor for early and late loss of islet mass is the critical lack of immediate vascularization and chronic hypo-oxygenation. Physiological supply of oxygen and nutrients in native islets is maintained by a tight capillary network, destroyed by the islet isolation procedure, restricting supply to diffusion from the portal vein and hepatic arterial capillaries until the revascularization process is completed. Oxygen tension in the liver parenchyma decreases from approximately 40 to 5 mmHg, eight-fold lower compared to the intra-pancreatic levels, leading to severe hypoxia, and β-cell death. Revascularization of the islet graft in rodent transplant requires 10-14 days and much longer in non-human primates and human recipients. Even after the revascularization of the islets is completed, the capillary’ density is significantly lower compared to the physiological intra-pancreatic situation. Finally, one of the main challenges is the cost of the procedure and some regulatory issues, as recently demonstrated by the ongoing discussion in USA (202). In fact, on April 15, the FDA's Cellular, Tissue, and Gene Therapies Advisory Committee voted in favor of approval of the biologics license application (BLA) seeking to market allogeneic islets of Langerhans for the treatment of ‘brittle” type I diabetes mellitus in adults whose symptoms are not well controlled despite intensive insulin therapy. The FDA endorsement of islet transplantation adds to the list of national agencies in Europe, such as the Federal Office of Public Health in Switzerland, the National Health Service (NHS) in the UK, the Swedish Local Authorities and Regions, the Ministry of Health in Poland and Belgium and, more recently, the French National Authority for Health (HAS) in France that have approved islet transplantation as a reimbursed standard-of-care procedure. Unfortunately, the FDA has chosen to consider islets as a biologic that requires licensure, making the universal implementation of the procedure in the clinic very challenging.

Table 8. Current Challenges Faced for Islet Transplantation

Challenge

Possible impact

Potential solutions

Progressive graft dysfunction

Reintroduction of exogenous insulin;

Destabilization of metabolic control;

Supplemental islet transplant.

Incretin mimetics;

Alternative islet implantation sites;

Novel immunosuppressive protocols.

Multiple islet donors

Increased operational costs;

Shortage of deceased donor pancreata for transplantation;

Risk of allosensitization.

Improved donor selection criteria;

Optimized cell processing;

Alternative sources of transplantable tissue (i.e., stem cells-derived or xenogeneic islets;

Alternative implantation sites.

Chronic immunosuppression

Systemic toxicity;

Increased risk of opportunistic infections;

Islet cell toxicity.

Use of biologics;

Immune isolation techniques; Development of immune tolerance inducing protocols.

Allosensitization

Reduced graft survival;

Preclude/worsen outcome of subsequent transplantation (i.e., islet or renal)

Maximizing the success rate of single donor islet transplantation;

Alternative sources of transplantable tissue;

Immune isolation;

Plasmapheresis / depletion of alloantibodies;

Novel immunosuppressive protocols;

Development of immune tolerance inducing protocols.

Cumbersome graft monitoring

Mainly rely on metabolic function tests, but cannot discriminate between immunological and metabolic causes of dysfunction;

Liver needle biopsies do not provide adequate graft tissue;

MRI and PET lack the resolution to detect islets scattered throughout the liver.

Improved simple metabolic measures predictive of graft dysfunction;

Improved sensitivity of noninvasive imaging techniques (functional MRI?);

Improved immune monitoring techniques for early detection of immune events able to discriminate between rejection and autoimmunity.

FUTURE DEVELOPMENTS IN BETA-CELL REPLACEMENT THERAPIES

The field of cellular therapies for the treatment of diabetes is rapidly evolving and a new exciting era has already begun (shown in Fig. 1). Efforts are ongoing to push to a broader dimension islet transplantation (143), including: (i) implementation of a scheme for donor and recipient selection and organ allocation to increase pancreas utilization (203-205); (ii) improvement and standardization of islet isolation process and its best codification by regulatory bodies (206-209) (iii) monitoring of transplanted islets by noninvasive imaging techniques (210,211); (iv) development of biomarkers to assess the efficacy of the immunosuppression/immunomodulation strategies (69,212-216); (v) identification of alternative transplantation sites (51); (vi) creation of an ideal bio-artificial niche for islet survival by bioengineering approaches (217,218) and (vii) use of immune-isolation techniques, such as hydrogel polymers that shield pancreatic islet from immune cell attack (219,220). On the other hand, there is increasing new excitement for the use of unlimited alternative sources of transplantable islets, such as xenogeneic (i.e., obtained from other species such as porcine islets) [reviewed in (221)] or derived from human stem cells (222-227). Pig islets may be available in plentiful amounts. Importantly, the ability to obtain genetically modified pigs that lack or overexpress specific molecules may be of assistance in developing cellular products with reduced immunogenicity for transplantation into humans. In turn, this technology may allow achieving long-term function under immunosuppressive regimens that are used for allogeneic cells or facilitating the induction of long-term acceptance of xenogeneic islet cells. Another area reporting great progress is that of regenerative medicine using human stem cells from embryonic or adult sources.

While adult stem cells, such as mesenchymal stem cells, have an immunomodulatory potential when infused at disease onset (228) (229) or as adjuvants to improve the outcomes of islet transplantation (230), the greatest enthusiasm lies in the possibility to use pluripotent stem cells to overcome the limits of islet transplantation (231,232). Human pluripotent stem cells (both embryonic stem [ES] and induced pluripotent stem [iPS] cells), are the best candidate for making β cells as they have unlimited potential for division and differentiation. Efficient protocols for the differentiation of pluripotent cells into β cells have been developed by several laboratories (233-241) and a great effort in the last year was concentrated on developing cellular products with consistent potency and safety profile for clinical application. Actually, six clinical trials using human pluripotent stem cells for the therapy of type 1 diabetes are registered in ClinicalTrial.gov: three active and recruiting (NCT03163511; NCT04678557; NCT04786262), one completed (NCT03162926), one enrolling by invitation (NCT02939118) and one active but not recruiting (NCT02239354). All these trials, except the NCT04786262, are using PEC-01 cells as a cell product. PEC-01 cells are a mixed cell population comprising pancreatic endoderm and poly-hormonal endocrine cells (233,242,243)differentiated by a pluripotent stem cell line called CyT49 (225). These pancreatic precursor cells are fully committed to further differentiate into mature endocrine pancreatic cells after their implantation and were tested within an encapsulation device in subcutaneous space. In December 2021, the interim results of some of these clinical trial appeared in two articles (244) . Over the follow-up period, which lasted up to 1 year, patients had 20% reduced insulin requirements, spent 13% more time in target blood glucose range, had stable average HbA1c <7.0%, had improved hypoglycemic awareness (average Clarke score decreased ∼1 point) associated with C-peptide levels that were, on average, ∼1/100th normal levels. Explanted grafts contained heterogeneous composition of pancreatic cells, including cells with mature β cell phenotype. In both the papers: (i) induction and maintenance immunosuppression appeared to be effective in preventing allogeneic and autoimmune destruction of the graft cells, (ii) the cell product appeared to be safe and well tolerated, since no teratoma formation was observed and the great majority of mild-to-moderate adverse effects was due to surgical procedure risks and side-effects of immunosuppression. These initial data reinforce the hope that pluripotent stem cells, differentiated into pancreatic endocrine cells, may be a renewable source of β cells for patients with T1D.

VX-880 is a second cell product approved in 2021 as investigational cell therapy for the treatment of type 1 diabetes. VX-880 consists of fully differentiated insulin-producing pancreatic islet cells obtained from pluripotent stem cells. A Phase 1/2, single-arm, open-label clinical trial was recently approved in patients who have T1D with impaired hypoglycemic awareness and severe hypoglycemia. VX-880 is infused in the portal vein and a chronic administration of concomitant immunosuppressive therapy is be required to protect the islet cells from immune rejection. Some preliminary results have already been shared in a press release in May 2022 and suggest that β cells fully differentiated from stem cells and transplanted into the liver may engraft and start secreting insulin early after infusion. In addition to ongoing clinical experiences, others commercial or academic organizations have announced their intention to conduct clinical trials of functional stem cell derived-islets (135). At this point, the need to shield the transplanted stem-cell-derived β-cells from immune rejection becomes more and more critical. In this direction, different strategies to reduce or avoid immune rejection are under evaluation (245) including (i) generation of universally compatible pluripotent stem cells by silencing or deleting HLA or genes essential for HLA expression or function and by expressing genes encoding immunosuppressive molecules (246), (ii) development of mild immunosuppressive regimens (e.g., monoclonal antibodies targeting NK cells and/or T cell subsets) sufficient to induce tolerance, (iii) improvement in encapsulation/containment of cell product and (iv) creation of a haplobank of stem cell lines (247).

A current limitation for islet transplantation is the inability to use non- or minimally-invasive predictive tests as well as biomarkers of early graft dysfunction to guide timely interventions aimed at preserving functional islet cell mass. Metabolic tests (i.e., glycemic control, insulin requirement, HbA1c, basal and stimulated c-peptide) remain the main indicators of graft function, the alteration of which may indicate underlying distress of the graft but cannot discriminate possible causes such as metabolic overload, immunity, or drug toxicity. In some cases, graft dysfunction may be reversible (i.e., transient metabolic overload due to an infection episode), but in many other cases at the time graft dysfunction is detected, a considerable mass of functional beta cells might already be irreversibly lost. Monitoring of transplanted islets by noninvasive imaging techniques (such as MRI, PET-CT, and US) is cumbersome, as they lack the resolution for the detection of cellular clusters the size of islets (~50-900um) that are scattered throughout the recipient’s liver [reviewed in (248)]. While encouraging preliminary studies have shown that preloading of aliquots of the islet graft with iron nanoparticles (for MRI) (249-253) or labeled glucose (for PET-CT) (254-257) can be used safely, these techniques do not allow assessing the whole mass of transplanted clusters and provide only passive and transient information on islet distribution in the transplant site. The progress in the field of functional MRI (fMRI) and toward the development of more sensitive beta-cell specific imaging techniques may allow a more objective assessment of islet cell mass over time in a near future. Detection of biomarkers [reviewed in (109)] in blood samples to determine immune cell function (i.e., cell surface expression of specific markers by flow cytometry and cytotoxic lymphocyte gene expression profiles, amongst other)(258-260) and autoimmunity reactivation (namely, autoantibody titers) is evaluated in ongoing clinical trials to identify means of assessing the efficacy of the immunomodulation strategies, detecting rejection episodes and reactivation of autoimmunity early enough to implement timely immune interventions to prevent graft loss (69,70,261-263). Unfortunately, some of the current tests lack adequate specificity as they may be affected also with underlying infections. With the rapid evolution of high throughput arrays, it is likely that new and more specific molecular biomarkers of islet cell distress and immune cell function will become available in the near future. Alternative transplantation sites [reviewed in (51)] are being currently explored that may contribute enhancing islet engraftment and attain sustained graft function long-term (52). Importantly, alternative sites may be modified using bioengineering approaches that could enable creating an ideal bio artificial endocrine pancreas [reviewed in (264)]. The use of immunoisolation techniques, such as using hydrogel polymers that shield islet cell clusters from immune cell attack, may contribute to achieve sustained function of transplanted cells without the need for life-long immunosuppression [reviewed in (265) and (264)].

CONCLUSIONS

In conclusion, islet transplantation as it is today cannot be the universal cure for type 1 diabetes. It represents a clinical option in few highly selected patients but it is the proof of principle that it is possible to replace efficiently β cells in patients with diabetes by a cell therapy. Restoration of physiologic metabolic control in patients with diabetes is highly desirable. Transplantation of islets of Langerhans allows the achievement of stable metabolic control in the most severe manifestations that cannot be matched with conventional medical therapies. The steady progress of clinical islet transplantation and the promising emerging new approaches that address immunity and beta cell sources justifies cautious optimism for the potential applicable of beta-cell replacement to all cases of insulin-dependent diabetes in the near future.

ACKNOWLEDGMENTS

This work was partially supported by the Italian Minister of Health (Ricerca Finalizzata RF-2009-1483387, RF-2009-1469691), Ministry of Education, University and Research (PRIN 2008, prot. 2008AFA7LC), Associazione Italiana per la Ricerca sul Cancro (AIRC, bando 5 x 1,000 N_12182 and Progetto IGN_ 11783), EU (HEALTH-F5-2009-241883-BetaCellTherapy) and the Juvenile Diabetes Research Foundation International.

The author alone is responsible for reporting and interpreting these data; the views expressed herein are those of the author and not necessarily those of the funding agencies.

ONLINE RESOURCES ON THE SUBJECT

Clinical Islet Transplant Consortium; Collaborative Islet Transplant Registry; Diabetes Research Institute Foundation; Health Resources and Services Administration; International Pancreas & Islet Transplant Association; The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); Organ Procurement and Transplantation Network; The Cell Transplant Society; Scientific Registry of Transplant Recipients; United States Department of Health and Human Services; United Network For Organ Sharing (UNOS).

American Diabetes Association; American Society of Transplantation; American Society of Transplant Surgeons; Beta Cell Biology Consortium; European Pancreas Club; European Society for Organ Transplantation; International Pancreas Transplant Registry; International Xenotransplantation Association; Juvenile Diabetes Research Foundation.

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Acromegaly

ABSTRACT

Acromegaly is a rare condition with an approximate incidence of 3-11 new cases per million of population per year and a prevalence of approximately 60 per million (1). There are approximately 3000 identified individuals in the UK and 15000 in the USA, although it is possible that more cases exist but do not come to clinical attention. More recent studies suggest a higher incidence of acromegaly, up to 6.9 per 100,000 according to Italian data and 7.7 patients per million per year in Iceland (2,3). The condition was named by Pierre Marie in 1886 using the Greek words akron- extremities and megas- large to describe the typical clinical appearance of the condition (4).The disease occurs as a result of excessive secretion of growth hormone. In more than 99% of cases this is due to a benign pituitary growth hormone secreting adenoma. Pituitary carcinomas are exceedingly rare. Extremely infrequently acromegaly occurs as a result of ectopic secretion of growth hormone releasing hormone (GHRH) from a peripheral neuroendocrine tumor (5,6), excessive hypothalamic GHRH secretion (7), or can result after long term exogenous GH abuse (8). Approximately 5% of cases are associated with familial syndromes, most commonly multiple endocrine neoplasia type 1 (MEN1) syndrome, but also McCune Albright syndrome, familial acromegaly, Carney syndrome, and Familial Isolated Pituitary Adenoma (FIPA). Both genders are equally affected and the diagnosis is typically made in adults aged 40-60 years of age. Younger patients often have more aggressive disease due to more rapidly growing adenomas. Acromegaly is associated with multiple systemic complications and a higher risk of mortality if untreated. Very often a multi-modal treatment approach is required to manage the condition, including surgery, radiotherapy, somatostatin analogues, GH receptor antagonist, and dopamine agonist. The management should be individualized to the patient using best practice guidelines, clinical experience, and individual patient circumstances and guided by biomarkers and clinical predictors.

PHYSIOLOGY: GROWTH HORMONE- STRUCTURE AND PHYSIOLOGY

Growth hormone is a 191 amino acid single chain protein containing two disulphide bonds. It has considerable structural homology with prolactin. Approximately 70% circulates as a 22 kD protein, 10% as a 20 kD isoform and the remainder as dimers or sulphated and glycosylated isoforms (Figure 1). Growth hormone secretion occurs in pulsatile bursts, numbering between 4 and 11 in 24 hours, especially at night, with extremely low or undetectable levels occurring in the nadir between pulses. Thus, a random single serum measurement is very limited as a means of assessing the overall level of secretion. Secretion of growth hormone is governed by both secretory and inhibitory hypothalamic factors. GHRH (growth hormone releasing hormone), ghrelin, and klotho act to stimulate release (9), whereas hypothalamic somatostatin (a 14 amino acid peptide) exerts marked inhibitory effects on GH release. Cortistatin has been found to exert dual, stimulatory and inhibitory effects on GH secretion (10). These stimulatory and inhibitory factors are subject not only to higher influences within the brain but also to peripheral signals such that the overall secretion of growth hormone can vary widely under different physiological and pathological conditions (11) . These are summarized in Table 1.

Table 1. Factors Affecting Growth Hormone Secretion (12)
PHYSIOLOGICAL		PATHOLOGICAL
Factors which increase GH secretion	Factors which reduce GH levels	Factors which increase GH secretion	Factors which reduce GH levels
Sleep	Overeating	Acute glucocorticoid excess	Chronic excess Cortisol/ glucocorticoids
Fasting	Obesity	Type 1 DM	Hyperthyroidism
Stress	Aging	Renal failure	Type 2 DM
Exercise	Increased IGF-1	Acute use of opiods
Hypoglycemia		Anorexia
Dopamine		Depression
Increased Amino acids i.e., high protein meal		Cirrhosis
Reduced Free Fatty acids
Glucagon
Testosterone and estradiol

Figure 1. The 2-D structure of human growth hormone.

Growth hormone circulates in blood bound to a specific binding protein, called GH binding-protein (GHBP). This protein comprises the extracellular portion of the growth hormone receptor (GHR), which is widely distributed and present in most tissues. Activation of the growth hormone receptor occurs when the growth hormone molecule binds two adjacent receptors resulting in dimerization of the growth hormone receptors. Dimerization of the growth hormone receptor results in its activation and binding of the intracellular Janus kinase (Jak 2) tyrosine kinase. The activated JAK2-GHR complex induces multiple signaling pathways responsible for the diverse actions of GH (13,14). These include phosphorylation of a) signal transduction and activators of transcription (STAT) proteins STAT1, STAT3 and most importantly STAT 5, b) SRC family kinases which trigger the MAP kinase pathway, c) insulin receptor substrate (IRS) proteins which activate phosphatidylinositol-3-kinase (PI3K) and Akt pathway, and d) SH2B1, a scaffold protein that upregulates GH action in the actin cytoskeleton (13). Intracellular growth hormone signaling is suppressed by several proteins, especially the suppressors of cytokine signaling (SOCS) 1-3 and protein tyrosine phosphatases SHP1, SHP2 (14,15). While there have been suggestions that GHR polymorphism could play a role in variable responses to the GHR antagonist Pegvisomant therapy, so far studies have not convincingly demonstrated this relationship (16).

Figure 2. The growth hormone molecule binding to the membrane surface growth hormone receptor. Signaling and transduction only occur when adjacent receptors bind the two specific binding sites on the growth hormone moiety to form a dimer.

One of the major proteins induced by growth hormone is insulin-like growth factor-1 (IGF-1) Although classical endocrinology states that it is hepatic derived IGF-1 acting in an endocrine manner that is responsible for most, if not all, of the effects of growth hormone, it is becoming increasingly clear that local production of IGF-1 acting either in a paracrine (nearby cells) or autocrine (on the same cell) manner also has important biological effects, predominant of which is stimulating cell proliferation and inhibiting apoptosis (17). Elegant gene 'knock-out' experiments have demonstrated that animals with selective hepatic IGF-1 loss have a normal phenotype and growth, despite marked reduction in serum IGF-1 levels (18). Furthermore, patients with severe GH deficiency, perhaps as a result of pituitary surgery, usually have serum IGF-1 levels just below or at the lower end of the normal range. Thus, rather than being the sole effector of growth hormone, serum IGF-1 should perhaps be more accurately regarded as a marker of serum growth hormone concentrations. Circulating IGF-1 does however have important effects in regulating pulsatile growth hormone secretion with IGF-1 acting in a negative feedback fashion suppressing growth hormone release.

Central Regulation of GH Secretion

GHRH consists of 44 amino acids, the first 27 from the N-terminus being essential for physiological activity (19). GHRH containing neurons are located in the arcuate nucleus and surrounding the venteromedial nucleus which is considered the major site of GHRH activity. Somatostatin producing neurons are predominantly present in the dorsolateral arcuate nucleus and share close synaptic connections with the GHRH neurons (20). While these two hormones form the key components of local autocrine short feedback regulation of GH secretion; it is further modulated by interactions with central neuropeptides. Dopamine, serotonin, norepinephrine, neuropeptide Y regulate GH output through their interactions with GHRH and Somatostatin neurons (20). GH exerts its own local negative feedback along with local IGF-1 feedback. The Neuropeptide Y system contributes to the rise in circulating GH seen in the fasting state (21).

A long negative feedback also exists and growth hormone induces hepatic secretion of IGF-1, which in turn inhibits growth hormone release through various mechanisms including modulating GH gene transcription, reducing GH mRNA expression through POUF1 /CREB protein interactions and altering somatostatin release through paracrine activity (20,22,23).

Ghrelin and Growth Hormone Secretion

GH pulsatility is primarily driven by nutritional status. The potent growth hormone stimulating action of ghrelin is now well established. Ghrelin was identified as a natural ligand responsible for regulating GH secretion in the acylated form (24). It is a 28 amino acid peptide, which is modified by Ghrelin-O-Acyl-transferase (GOAT) enzyme to mediate its GH secretory action through IP3 signaling pathway (25). Expression of ghrelin is found in many tissues including both the gastrointestinal tract and the CNS, with the strongest concentrations located in the stomach. Ghrelin mediates its orexigenic actions through the vagal nerve stimulation and eventually acting on the hypothalamic appetite center though the noradrenergic pathway (26). Gastric expression of ghrelin is reduced following feeding and increased by fasting, hypoglycemia and after leptin administration. On other hand, leptin seems to variably effect GH secretion depending on the nutritional status. The exact mechanisms of leptin driven GH changes are not well understood (27).

Figure 3. The growth hormone/ insulin-like growth factor-I axis.

Peripheral Regulation of GH Secretion

Systemic energy homeostasis is a potent influence on GH secretion, with interactions by glucose, free fatty acids, adipokines, leptin, ghrelin, and insulin (28-30). Several hormone systems have regulatory effects on growth hormone secretion. Hypothyroidism is associated with low levels of both growth hormone and IGF-1, and in children leads to short stature (31). Thyroxine replacement has been shown to reverse these deficits. Further evidence from studies in rodents indicates that growth hormone gene expression is regulated by thyroid hormone acting through a thyroid hormone responsive element in the promoter region of the growth hormone gene (32,33). GH replacement has been shown to reduce the Free T4 levels and increase Free T3 levels, by the effect on type 2 iodothyronine deiodinase (34). Glucocorticoids are inhibitors of somatic growth both in humans and experimental animals and individuals with either Cushing’s syndrome or taking exogenous corticosteroids have been shown to have reduced growth hormone secretion.

Gonadal hormones also play a role in the neuroregulation of growth hormone secretion. In both sexes spontaneous growth hormone secretion is increased during puberty, and reduced in those with delayed puberty (35), suggesting that both estrogen and testosterone influence growth hormone secretion. The estrogen related reduction of IGF-1 seems to be responsible for relatively higher GH levels in females compared to men, with similar IGF-1 levels (36,37). Hypoglycemia is a potent inducer of growth hormone secretion, and insulin induced hypoglycemia remains the most employed provocative test of growth hormone reserve in humans. Hypoglycemia reduces hypothalamic somatostatin secretion facilitating growth hormone release. A delayed rise in GH levels is noted after acute hyperglycemia and is likely a result of GHRH rise (38) In contrast, hyperglycemia suppresses growth hormone secretion from the healthy pituitary. The availability of amino acids as in the post-prandial state stimulates growth hormone secretion whilst elevated non-esterified fatty acid levels suppress growth hormone release.

IGF-1: Structure and Function

IGF-1 is a single chain polypeptide of 70 amino acids with three intrachain disulphide bridges, coded by a gene situated on the long arm of chromosome (39). It has 48% amino acid sequence homology to pro-insulin, the A and B domains of IGF-1 have 60-70% homology but there is no homology with the C domain. IGF-1 has a specific receptor, which is structurally and functionally very similar to the insulin receptor. It consists of two extracellular α-subunits which are the hormone binding sites and two transmembrane β-subunits which are involved in initiating intracellular signaling. Post-receptor signaling mechanisms are also similar for IGF-1 and insulin receptors, both activating the tyrosine kinase and IRS-1 cascades. IGF-1 can bind to the insulin receptor but with only 1-5% affinity compared to insulin. Under normal physiological conditions it is thought that IGF-1 acts via the specific IGF-1 receptor, but in the presence of high concentrations of IGF-1 there is likely to be cross activation with the insulin receptor. IGF-1 receptors are found on most tissues with the notable exceptions of liver and adipose tissue. Hybrid IGF-1/insulin receptors have now been well documented and sequenced but their role is unclear (40).

The majority of circulating IGF-1 is produced by the liver with bone, adipose tissue, kidney, muscle and many other tissues producing a smaller quantity. Plasma concentrations of IGF-1 in the human are regulated by growth hormone, insulin, age, and nutritional state. Bioavailability of IGF-1 is determined by its binding proteins (see below). Growth hormone and insulin are the main regulators of hepatic IGF-1 production. The precise regulation of local IGF-1 synthesis is uncertain, but it is influenced by many other trophic hormones such as ACTH, fibroblast growth factor, and TSH (41).

Figure 4. The regulation of growth hormone secretion.

IGF Binding Proteins (IGFBPs)

Unlike insulin the majority of IGF-1 circulates in plasma bound to a variety of binding proteins which determine its bioavailability and modulate its biological action (42). The majority of IGF-1 is bound in a 150 KDa complex with IGFBP-3 and form an acid labile subunit ALS (42). This large molecule (termed the ternary complex) is unable to pass through endothelium and acts as an intravascular reservoir of inactive IGF-1. The half-life of IGF-1 in the complex with IGFBP-3 and ALS is 12-15 hours compared with 10-12 minutes for free IGF-1. The exact mechanisms by which IGF-1 is released from the ternary complex to allow access into the tissues is not known; however, IGFBP degrading protease activity has been well documented in many biological fluids and clinical states.

Current knowledge suggests that IGFBP-1 and IGFBP-3/ALS are the binding proteins which have the major effects on the bioavailability of IGF-1(40). IGFBP-1 is inversely related to insulin levels, has a circadian variation with the highest levels being found overnight when insulin levels are lowest, and inhibits the hypoglycemic action of IGF-1 (43). Growth hormone secretory status is the main regulator of plasma levels of ALS (42).

PATHOLOGY OF ACROMEGALY

Acromegaly is most commonly the result of pituitary adenoma and rarely due to non-pituitary neuroendocrine tumor/ neoplasia (NET/ NEN). Pituitary tumors are commonly monohormonal or plurihormonal in nature with further distinct subtypes. These subtypes of tumors are responsible for their characteristic behavior and can sometimes guide management (44). The common subtypes are discussed below.

1) Monohormonal densely granulated somatotroph adenomas are the most common type of GH-secreting pituitary tumors. They have predominance of the large, dense secretory granules which contain GH and appear deeply eosinophilic on staining. They account for 30-40% tumors and while tend to lead to higher GH levels, they are usually slow growing tumors, in older patients with mild disease, and retain a predictable response to SSA therapy. Somatic mutation in Gs-α subunit of GNAS has found to be the most common abnormality in GH secreting tumors leading to increased cAMP activity (45).

2) Sparsely granulated somatotroph adenomas form the second most common type of tumor. As the name suggests, they are lightly eosinophilic on HE staining due to the increased presence of keratin aggregates and reduced GH containing granules. While they present with lower GH levels, the tumor behavior is relatively aggressive. They tend to be larger, more invasive with higher ki67 proliferation indices and are less responsive to SSA therapy. Further studies suggest reduced expression of E cadherin and SSTR2 in sparsely granulated tumors as factors likely to be responsible for poor response to SSA (44).

3) Mammosomatotroph adenoma from a monohormonal Pit-1 lineage cells is a common pathology found in younger individuals with acromegaly. They are densely granulated and co-stain for GH and PRL. They behave in a more benign fashion with high GH and PRL levels leading to early presentation, when the tumor is smaller at diagnosis. They respond to SSA therapy similarly to densely granulated tumors (46).

4) Mixed somatotroph and lactotroph tumors are formed of bihormonal cells with variable combination of somatrotroph and lactotrophs. In variable combinations they comprise of sparsely granulated and densely granulated cells all of which express Pit 1. They tend to be less amenable to treatment and are reported to have frequent disease recurrence (47).

5) Mature Plurihormomal Pit1-Lineage tumors frequently immunostain for TSH along with GH, PRL and are found to express GATA3 (48). Clinical presentation includes features of thyroid overactivity with thyrotoxicosis with non-suppressed TSH.

6) Acidophil stem cell adenomata are tumors comprising of immature GH and PRL secreting tumor cells of a single precursor. The histology shows chromophobic or slightly acidophilic cells, with abundant granular cytoplasm of oncocytic distribution (44). Patients present with hyperprolactinemia which is disproportionate to the size of the tumor. They are frequently invasive and less responsive to dopamine agonist therapy.

7) Poorly differentiated Pit-1 lineage tumors comprise of tumor cells with strong expression of Pit-1 and variable expression of estrogen and GATA3. They are polyglonal and spindle shaped poorly differentiated cells which stain variably for GH, PRL, TSH, and alpha subunit. They are usually macroadenomas, with invasion of surrounding structures and high risk of recurrence (46).

8) Pituitary carcinomas are very rare and form less than 1% of cases. They are difficult to distinguish clinically, and diagnosis is confirmed with evidence of distant metastasis and high ki67 index (e.g., >10%) on histology. These tumors frequently require multi-modal therapy, including chemotherapy, temozolamide and radiation (44).

9) Pituitary hyperplasia is suspected radiologically when there is a uniformly enlarged pituitary gland with no distinct focus of gadolinium enhancement. Hyperplasia is confirmed on histology when the pathology shows expanded pituitary acini containing all of the adenohypophysial cell types, but with increased numbers of somatotrophs and/or mammosomatotrophs (46). The histological diagnosis should prompt the clinician to explore for a GHRH secreting tumor elsewhere or consider investigations for specific genetic conditions associated with acromegaly (highlighted in table 4) such as MEN1, Carney Complex, and McCune Albright Syndrome (49).

Non-pituitary sources of disease include GHRH or GH secreting central and peripheral tumors. Hypothalamic tumors such as hamartomas, choristomas, gliomas, and gangliocytomas producing GHRH result in pituitary hyperplasia and very often the diagnosis remains elusive until patient has undergone pituitary surgery. Carcinoid tumors secreting GHRH are recognized as rare peripheral cause of acromegaly and usually are bronchial in origin (50). An ectopic location of pituitary adenomas has been reported in the tract of dorsal migration of the adenohypophysial cells. While a significant number of peripheral tissues have been found to secrete GH (51), reports of GH secreting peripheral tumors include lung, pancreatic and adrenal tumors.

Table 2. Pathology Associated with Acromegaly (44,52-54)
Pituitary adenoma	Densely granulated somatotroph adenoma Sparsely granulated somatotroph adenoma Mixed cell somatotroph and lactrotroph adenoma Mammosomatrotroph (monohormonal Pit-1 lineage) adenoma Acidophil stem cell adenoma Plurihormonal adenoma Poorly differentiated Pit-1lineage tumor Pituitary hyperplasia Pituitary carcinoma Rare: ectopic pituitary adenomas identified in sphenoid sinus or parapharyngeal tissue
Ectopic hormone secretion	Central: Hypothalamic tumors Ganglioneuroma Peripheral: Bronchial carcinoid, small cell lung cancer, adrenal tumor, pancreatic neuroendocrine tumor
Exogenous GH replacement or abuse
Pseudoacromegaly	Pachydermoperiostosis IGF signaling pathway diseases Severe insulin resistance

CLINICAL FEATURES OF ACROMEGALY

The clinical manifestations of acromegaly evolve gradually over a long time and as a consequence there is a lag time of about 5-10 years, from symptom onset to diagnosis (55,56). In the recent decades, there seems to be some early recognition of the condition, particular in individuals investigated for pituitary ‘incidentaloma’, for example with hypogonadism as a presenting feature (57). One third of the cases, have co-existent symptoms of hyperprolactinemia, which aids in early diagnosis (58).

Growth hormone secreting pituitary adenomas are frequently (more than 70%) large tumors (macroadenoma, ≥ 10 mm in diameter) which may present with local mass effects such as headache (often severe and out of proportion to the size of the pituitary tumor), hydrocephalus, visual field defects, ophthalmoplegia, or other cranial nerve palsies (59). As the lesion increases in size deficiencies of other anterior pituitary hormones may also occur. Microadenomas (< 10 mm in diameter) are conventionally thought to be less common, but tend to represent one third of the cases (60,61). However patients presenting with pituitary tumors, without clear features of acromegaly may have elevated IGF-1, and GH positive immunohistochemistry on the resected tumor specimen (62) and such silent growth hormone tumors seem to be more common in females with a higher risk of recurrence (63). The term micromegaly is used to describe such clinical presentations (64,65). Recognition of such presentations should prompt the endocrine specialist to consider GH secreting tumors in all presentations, but especially in the younger patient with pituitary tumor.

Hypopituitarism has been found to occur in about 40% cases with variable frequency of hypogonadism, adrenal insufficiency, and secondary hypothyroidism (66,67). Hypogonadism, presenting as decreased libido, infertility or oligo/amenorrhea is a common finding at presentation; it may be due to both gonadotrophin deficiency as well as hyperprolactinemia, either from coexistent excessive secretion of prolactin or from stalk compression. Hypogonadism has been reported even in patients with microadenomas with normal prolactin, thereby suggesting an independent effect of GH hypersecretion (68). Menstrual irregularities, PCOS, subfertility and erectile dysfunction can occur as a consequence of GH excess (69,70). The occurrence of diabetes insipidus in relation to a pituitary adenoma is extremely rare and should raise the possibility of an invasive pathology (71,72).

Soft Tissue and Skeletal Changes

The most characteristic feature and one that usually precipitates the diagnosis is a change in appearance as a consequence of soft tissue and bony changes. The common changes include coarsening of the facial features, broadening of the nose, thickening of the lips, macroglossia, and prominence of the supraorbital ridges. There is enlargement of the hands resulting in their characteristic 'spade-like' appearance and soft dough-like consistency of the palms. Ring size increases; a sensitive objective assessment of disease activity and response to treatment. Similar changes occur in the feet which become wider with increase in shoe size. Elongation of the jaw results in prognathism which contributes to dental malocclusion, interdental separation, and temporomandibular joint pain (73,74).

Greasiness of the skin is a frequent finding with excessive sweating, one of the most sensitive signs of growth hormone excess. Skin tags are a frequent finding, likely related to epithelial cell hyperproliferation in response to IGF-1 (74). Additional dermatological manifestations include hypertrichosis, psoriasis, acanthosis nigricans, and cutis verticis gyrata, with the latter two seen more commonly in severe cases (75). Skin changes are a result of deposition of glycosaminoglycans in the subcutaneous tissue, along with increased proliferation of dermal fibroblasts as a consequence of GH and IGF-1 action (74). These changes tend to reverse after treatment, at least partially if not completely. The lean body mass is higher in individuals with acromegaly, while there is increase in adipose tissue post therapeutic intervention (76). This reversal of body composition tends to stabilize by three months of the surgery (77). In addition to the negative impact on body fat, reversal of GH excess has been found to increase intrahepatic lipid accumulation (78).

Generalized organomegaly is not well reported with acromegaly, in contrast some earlier assumptions of the disease process but enlargement of thyroid, prostate, salivary glands, heart, liver and spleen has been recognized (79). Macroglossia, increased thickness of laryngeal structures and vocal cord enlargement increases the risk of anesthesia and makes intubation difficult (80). Ultrasound evidence suggests the presence of increased organ stiffness and commonly reported features include renal cysts, thyroid nodules, multinodular goiter, gallbladder polyps, and polycystic ovaries (79). Mucosal edema and hypertrophy of vocal cord can result in voice changes, but true existence of voice abnormalities is debated (81,82). Patient reported questionnaire and evaluation of voice parameters seem to demonstrate presence of micro perturbations, lower amplitude and poor quality of voice in individuals with active disease (82,83).

The skeletal manifestations of acromegaly result from multiple factors which include direct effect of GH, IGF-1, altered calcium phosphate metabolism, hypogonadism, diabetes, and over replacement of steroids (84,85). Acromegaly results in increased bone resorption and altered bone formation according to various cross-sectional studies, but the effect of GH, IGF-1 on bone health is complex. GH mediates its effect on bone through systemic IGF-1 with action on cortical bone, whereas bone IGF-1 seems to be responsible for cancellous bone health. There is emerging evidence of correlation of sclerostin levels with acromegaly related bone disease (86). The high prevalence of vertebral fractures (VF) in active acromegaly has been known for a long time with some studies reporting incidence as high as 60%, and dependent on the duration of disease and gender (males>females) (84,87). In a French study, authors have suggested that the skeletal abnormalities are more likely vertebral deformities than true fractures (88). Screening for VF is recommended in all patients with active acromegaly as it has a significant impact on quality of life, morbidity, and development of cardiac and pulmonary complications (85,89). Standard DXA seems to be less reliable in predicting the risk and Volumetric DXA with quantitative assessment of the trabecular bone density seems to provide more reliable information of acromegaly related bone disease (87,90). Unfortunately it is difficult to undertake this test routinely and therefore alternative use of newer methods such as non-invasive 3D-SHAPER and TBS Trabecular bone score assessments may be considered (90). While vertebral changes are most discussed, consequences of acromegaly include development degenerative diseases in all weight bearing and non- weight bearing joints, predominantly shoulder, hip and knees (84,91). The prevalence of radiographic evidence of at least one joint involvement has been found to be as high as 99% (92). Patients with active disease seem to be have higher prevalence of reduced cortical density at hip compared to patients with non-functioning pituitary adenomas (93). Degenerative joint changes in early acromegaly related arthropathy appear different from usual osteoarthritis, and are noted as widened joint spaces contributed by cartilage hypertrophy and marked osteophytosis, in contrast to standard OA (94). Acromegaly related arthropathy is associated with a significant impact on quality of life and tends to progress despite improvement in disease status (89). Routine use of anti-resorptive therapy is currently not well established, but considering the wider implications of the disease on bone health in active disease, it seems reasonable to consider offering bone protective therapy in patients with early evidence of bone disease (90).

Figure 5. The typical facial appearance of acromegaly. Evolution of the appearances over 2 decades

Sleep Disordered Breathing

Sleep apnea syndrome (SAS) is a well-recognized manifestation of GH excess and seems to have been variably reported with incidences of 40-80% and about 11.7-20 times higher prevalence than in general population (55,95). Evaluation of the bony changes in the facial skeleton showed significant differences in patients with acromegaly and SAS, compared to patients without SAS. But soft tissue enlargement of upper airways contribute more to the narrowing of the pharyngeal airway space than the craniofacial skeleton changes (95). CT and MRI assessment of upper airways demonstrate pharyngeal hypertrophy and upper airway stenosis correlating with the severity of obstructive sleep apnea (96). Patients with SAS have been found to have more features consistent with metabolic syndrome, such as hypertension and DM compared to patients without SAS (97). Improvement in SAS noted after intervention seems to correlate with positive changes in tongue volume and pharyngeal soft tissue (98). Unfortunately, complete reversal of SAS does not occur and persistent SAS is seen in about 40% of treated cases. It is likely due to effect of additional factors on SAS such as age, male gender, smoking, and obesity (84). A direct inhibitory effect of GH on central respiratory center can result in a non-obstructive central pattern of sleep apnea (99). This phenomenon seems to be much less prevalent than obstructive disease, while a mixed pattern has also been reported (100).

Muscular Changes

Musculoskeletal pain is typically progressively evident during the disease course, with close to 90% patients reporting pain as a dominant symptom (101). Irrespective of the severity of the acromegaly, pain contributes significantly to reduced quality of life (101). Studies demonstrate that GH excess leads to hypertrophy of the type 1 muscle fibers with variable findings for type 2 fibers (102-104). The predominant abnormality of type 1 muscle fibers strength seems to be responsible for evident muscle weakness noted in high velocity activities (105). While earlier studies consistently reported muscle weakness in acromegaly, a recent study used quantitative measures suggest patients with active acromegaly may have higher proximal muscle strength, but reduced hand grip which normalizes after treatment (103). It is postulated that the difference in findings could be related to pain interfering with true assessment of muscle function (103). Ultrasound evidence showed increased tendon thickness, enthesisitis, soft tissue enlargement but reduced muscle volume in some lower limb muscles in a cohort of thirty nine patients (106). There is emerging evidence that acromegaly has a complex effect on muscle strength and volume (107). Differential interactions of GH, Muscle Ring Factor -1 (MuRF-1), and myostatin seem to be responsible for chronic effects of GH excess on skeletal muscles (108).

Neurological Abnormalities

Carpal tunnel syndrome is present in approximately 60% of patients at diagnosis but about 80% will have electrophysiological evidence of median nerve neuropathy (109). The pathophysiology is due to swelling of the median nerve itself within the carpal tunnel rather than extrinsic compression from increased volume of the carpal tunnel contents (110). This is well evident on MRI and Ultrasound studies of the median nerve, and seems to correlate with the abnormal nerve conduction studies (110-112). Similar peripheral nerve abnormalities and abnormal nerve conduction studies have been noted in other peripheral nerves including peroneal, tibial, ulnar, sural nerves (112,113)and polyneuropathy is more common in uncontrolled disease (114). Unfortunately it seems some of the changes are not completely reversible, despite normalization of disease markers (115). Patients with active acromegaly have altered cardiac autonomic function, which contributes to the cardiovascular risk (116,117). This seems to respond to active intervention (118). There is not much evidence of central nervous involvement related to the disease (113), with some report of delayed brainstem auditory evoked potentials (119). Restless leg syndrome has been reported to be present in 20% of cases and negatively impacts the quality of life (120).

Cardiac Complications

Cardiovascular diseases continue to remain one of the most common causes of morbidity in patients with acromegaly and account for 60% of the mortality with this condition (121,122). The range of abnormalities detected at the time of diagnosis have been reported to be hypertension, cardiac hypertrophy, arrhythmias, coronary artery disease, and systolic heart failure, in the order of prevalence (123,124). Hypertension presents with higher diastolic readings than systolic BP measurements (125). Based on ambulatory BP monitoring readings, it seems that the prevalence of hypertension is about 22% at diagnosis, much lower than earlier studies with single office measurements (126). Anti-natriuretic effect of GH is a direct consequence of GH action on the epithelial sodium channel ENaC in cortical collecting ducts of the kidney (127). This sodium retention leads to volume expansion and further compounded by GH effects on cardiac output, impaired endothelial function, increased peripheral resistance and co-existent sleep apnea are some factors that lead to development of hypertension (128).

Acromegaly related cardiomyopathy is a consequence of GH and IGF-1 effect on cardiac myocytes, regulation of cardiac muscle specific gene transcription, and increased fibrosis (129). It has been described to undergo three stages of disease progression (130). In the first phase biventricular concentric hypertrophy is described related to muscle hypertrophy and increased contractility and leads to a hyperkinetic syndrome. As the disease progresses, patients tend to develop diastolic dysfunction with more prominent ventricular hypertrophy. Patients report reduced exercise tolerance and it is common for disease to be diagnosed in this phase. If untreated patients may progress to develop overt diastolic and systolic dysfunction presenting as congestive heart failure in about 3-4% cases and is a poor prognostic marker (123). The presence of left ventricular hypertrophy correlates with disease duration and various studies report prevalence from 11-78% (84,131). Cardiac Magnetic Resonance (CMR) studies have demonstrated variable prevalence of left ventricular hypertrophy compared to echocardiographic studies (132,133). CMR is more reliable in identifying myocardial fibrosis and RV systolic dysfunction then echocardiography (133). Despite the presence of cardiovascular abnormalities associated with ischemic heart disease, recent studies report no increase in prevalence of Ischemic heart disease, in comparison to normal population (55,134).

Cardiac arrhythmias have been reported to occur in about 7-40% cases of acromegaly. A wide range of rhythm disturbances described in patients with acromegaly include paroxysmal atrial tachycardia, supraventricular tachycardia, sick sinus syndrome, ventricular ectopic, and ventricular tachycardia (130). A typical acromegaly related left ventricular rhythm disturbance, results from abnormal and dyssynchronous loss of peak contraction of corresponding cardiac segments (135). Mitral and aortic regurgitation have been commonly associated with acromegaly and seem to correlate with the duration of the disease (136). Unlike cardiomyopathy and arrhythmias which improve or even completely reverse with disease control, valvular disease is irreversible and only tends to stabilize with intervention (130).

Metabolic Complications

Growth hormone is a potent insulin antagonist and acromegaly results in abnormal glucose tolerance in many patients with frank diabetes mellitus in up to 50% cases at diagnosis. Lipid abnormalities, in particular elevation of serum triglycerides, reduced HDL levels, increased small dense LDL particles, and increased lipoprotein-a (Lp(a)) may be an accompanying feature of insulin resistance and is noted in one third of the cases (137,138). Chronic GH excess results development of insulin resistance by several mechanisms. Reduced glucose uptake occurs by increased levels of free fatty acids and reduced expression of GLUT1 and GLUT 4 receptors (139). GH also results in development of a pro inflammatory state in the adipose tissues with alterations of the genes coding visfatin and IL6 (140). The degree of insulin resistance correlates with IGF-1 levels (141) and improves with management of the disease. Development of IGF receptor resistance beyond a threshold for IGF-1 has been reported in states of chronic GH excess leading to further insulin resistance (142). Visfatin and irisin levels have been suggested to correlate with metabolic abnormalities and cardiovascular risk factors (143,144). When choosing treatment, octreotide, and lanreotide have less impact on the glycemic variations, while pasireotide can aggravate hyperglycemia. Pegvisomant has a favorable on the metabolic parameters (145).

Table 3. Clinical Manifestations and Complications Reported with Acromegaly
Tumor related local effects	Headache Visual field defects Cranial nerve abnormalities Hydrocephalus Temporal lobe epilepsy Hyperprolactinemia Hypopituitarism
Systemic effects
Skin changes	Hyperhidrosis Oily skin Skin tags Hypertrichosis Acanthosis Cutis verticis gyrata	Cardiac	HT Cardiomyopathy Valvular heart disease Arrhythmia Heart failure
Soft tissues changes	Acral enlargement Change in voice quality Visceromegaly (thyroid, prostate, liver, salivary glands)	Neurological	Peripheral nerve abnormalities Autonomic dysregulation Lumbar canal stenosis Narcolepsy Restless leg syndrome
Orofacial changes	Prognathism Frontal prominence Dental malocclusion TMJ pain Gingival enlargement Macroglossia	Pulmonary	Sleep apnea Restrictive lung disease Subclinical hypoxemia
Musculoskeletal	Vertebral deformities Kyphosis Arthralgia and arthritis Myopathy Degenerative arthropathy Calcific discopathy Hypermobility	Neoplastic	Colon polyps Thyroid cancer Breast cancer
Endocrine and Metabolic	Hypogonadism PCOS DM, insulin resistance Hypertriglyceridemia Erectile dysfunction	Renal	Increased GFR Hypercalciuria Glomerulosclerosis
Hematological	Increased thrombosis risk	Psychiatric	Depression
Ocular	Increased risk of diabetic retinopathy Extraocular myopathy Glaucoma Epiphora

Thyroid Abnormalities

Patients with GH excess have been demonstrated to have a rise in TSH and T3 levels with no significant relation with the FT4 levels (146). There is a direct correlation of IGF-1, GH levels with thyroid volume. Multinodular goiter is one of the most common thyroid abnormality in patients with acromegaly with frequencies of 69.5 to 79.1% being reported by some authors (147). Patients in remission after surgery have been shown to have change in the consistency of the thyroid nodules, reduced vascularity, and volume (148). Over the course of follow up of patients with active acromegaly, thyroid nodule enlargement seemed to correlate with IGF-1 levels with increased prevalence of differentiated thyroid cancer (papillary thyroid carcinoma) in this subgroup (149,150). Despite the increasing understanding, routine screening for thyroid nodules is not yet recommended but assessment should certainly be considered in patients with a palpable nodule (151).

Neoplasia

The true risk of cancer continues to remain debated with concerns of heterogeneity in the study population, selection biases, variable screening strategies, and limitations of using standardized incidence ratios as the reporting indices (84). In the recent years, some large cohort population studies suggest higher cancer risk than general population. Acromegaly has been associated increased risk of cancers, particularly colon, kidney, and thyroid cancer in a large Italian survey (152). A similarly large Danish cohort has reported increased incidence of colon, thyroid, breast, gastric, and urinary bladder cancers (153).

Animal models and in vitro studies suggest anti-apoptotic and tissue proliferative role of GH and IGF-1. IGF-1 deficiency has been found to result in protection from tumor development. GH signaling pathways and autocrine GH action contribute to tumorigenesis and in colonic tissue this mechanism results in reduced action of tumor suppressor proteins (84,154). Colon cancer has been studied in detail in patients with acromegaly. Patients have a higher prevalence of adenomatous and non-adenomatous polyps than general population (155) with reported trend varying between 6-30% (156). Colonic pathology is related to disease activity with patients with elevated serum growth hormone and IGF-1 levels being particularly prone to developing colonic adenomas (157). Although the exact pathogenesis of these tumors remains uncertain it is likely to involve altered homeostasis of cell numbers within the colonic epithelial crypts; increased proliferation and decreased apoptosis within the crypts of patients with acromegaly have both been documented (158). Colorectal neoplasia in acromegaly has different characteristics compared to the general population, in that the adenomas are more likely to be located in the right side of the colon, tend to be bigger and are more often multiple as well as demonstrating increased dysplasia (159).

It is now generally accepted that patients with acromegaly should be regarded as a high-risk group for colorectal cancer and regular colonoscopy screening should be offered to all patients. Current evidence suggests that this should begin at the age of 40 years with the subsequent interval depending both on disease activity and the findings at the original colonoscopy screening (160). In the presence of a polyp (hyperplastic or adenoma) or elevated serum IGF-1 levels screening should be repeated after five years, whilst a normal colonoscopy screening, or serum IGF-1 level within the normal range suggests screening every 10 years may be appropriate. As approximately 30% of lesions occur at the cecum or in the ascending colon, total full-length colonoscopy is required. This should be performed by an experienced colonoscopist, as the cecum is reached in only about 70% of patients in inexperienced hands. Due to their slow bowel transit time and elongated colon, patients with acromegaly require rigorous bowel preparation, often twice that necessary for the patient without acromegaly. Failure to visualize the cecum necessities a repeat colonoscopy or failing this examination using CT virtual colonoscopy (156).

Lung Complications

Pulmonary complications are common in acromegaly. Total lung volume and residual volume are increased, along with narrowing of both large upper airways and more commonly small airways (161,162). In a large study Storrmann et al reported higher prevalence of small airway obstruction in females. They also highlighted presence of subclinical hypoxemia in patients with acromegaly. The findings did not correlate with levels of IGF-1 of duration of disease (163). Tracheal structural abnormalities have been found to be responsible for large airway disease (164).

Ocular and Auditory Complications

A wide variety of ocular complications have been reported in patients with acromegaly, apart from visual field defects. The prevalence of proliferative diabetic retinopathy has been variably reported to be higher in patients with acromegaly (165) in some studies, while others suggest it is likely that there is increased retinal vessel branching, and noted no difference in retinopathy rates (166). Extraocular muscle enlargement has been reported by few authors and rarely has resulted in presentation of diplopia (167-169). Studies have highlighted increased intraocular pressure, increased corneal thickness and increased retinal thickness in patients with acromegaly, with rare reports of epiphora (170-172). The association of acromegaly with hearing disturbances has not been well reported. There have suggestions of abnormal bony changes, changes in middle ear pressures and internal acoustic meatus contributing to variable hearing abnormalities (173,174). But findings have not been widely validated and association of acromegaly with hearing loss is not well established (175).

Other Systemic Complications

Hematological abnormalities are not common with acromegaly. Recent evidence suggests patients with active acromegaly may be at a higher thrombotic risk and this could contribute to cardiovascular risk (176,177). Higher levels of fibrinogen, factor VIII and thrombin tend to result in hypercoagulable state in active untreated disease (178). Rare case reports of polycythemia, myeloma, and Waldenstrom’s macroglobulinemia have been reported (179,180).

GH and IGF-1 receptors have been found in kidneys and suggest local autocrine and endocrine activity of GH at the level of nephrons (51). The effect of GH excess on kidneys has not been well described. Chronic GH exposure leads to renal hypertrophy and structural changes to include glomerulosclerosis (181) . Patients with acromegaly have been reported to have increased GFR, reduced renal excretion of sodium, potassium, hypercalciuria, hyperphosphaturia, greater prevalence of microalbuminuria and micro-nephrolithiasis, irrespective of comorbidities (182-185).

Morbidity and Mortality in Acromegaly

It is established that uncontrolled acromegaly results in a considerable increase in morbidity with an overall mortality at least two-fold that of the general population (186). In early epidemiological reviews more than 50% of patients had died by the age of 60 years, usually as a result of diabetes, cardiovascular, respiratory or cerebrovascular disease (187). With improved treatment of both the underlying disease and these complications, patients are now surviving longer although may then be susceptible to other complications such as malignancy (188). The determinants of mortality included older age and IGF-1 levels at diagnosis, treatment modality, and malignancy (121). Prolonged diagnostic delay has been found to positively correlate with morbidity and mortality (189). Longitudinal population cohorts indicate a negative impact of female gender on the presence of comorbidities and mortality (37,190).

DIAGNOSIS OF ACROMEGALY

The diagnosis is made using a combination of clinical examination and biochemical assessment. Serum growth hormone concentrations are typically elevated, and although pulsatility may be reduced, levels may fluctuate widely in acromegaly. Due to the pulsatile nature of growth hormone secretion, a single growth hormone measurement is of little use in either monitoring or confirming the diagnosis of acromegaly. GH levels are affected by age, gender, and comorbid disease states and these factors need to be taken into account when interpreting results. These factors have been outlined in table 1. Due to variations between assays, it is recommended that a standardized and similar performing assay is used for monitoring of the disease activity. Most modern assays detect the highly prevalent GH 22kDa isoform and the most common preparation used to calibrate GH assays is the latest recombinant IRP 98/574 (197). It is useful to be aware of the interferences of the local assay with biotin and Pegvisomant. Various tests used in screening, diagnosis, and management of acromegaly are discussed below.

Biochemical Tests at Diagnosis

IGF-1 levels: A single serum IGF-1 level has been advocated as being a useful first line test for the diagnosis of acromegaly as it is elevated in the majority of subjects. It is an indirect assessment of growth hormone secretion with approximately 25% of patients having a discrepancy between the mean value of a growth hormone day curve and an IGF-1 level. IGF-1 secretion is subject to several influences including liver and renal dysfunction, nutrition, diabetes mellitus, physiological factors such as age, gender, and the presence of a statistical correlation between its levels and those of growth hormone should not be used as proof that they are interchangeable (198). However, despite these limitations, from a practical point of view, an elevated serum IGF-1 measurement may be useful as confirmatory evidence, assuming that age and sex matched normal ranges are used, and for monitoring treatment (199). Standardized IGF-1 (IS 02/254) is recommended for manufacturers of IGF-1 assays (52,200).
GH day curve: The assessment of growth hormone hypersecretion requires the mean value of serial samples taken throughout the day (e.g., 5 samples over a 12-hour period). The samples should be taken through an indwelling venous cannula to avoid the stress effects of repeated venipuncture. In normal subjects, the majority of values throughout the day are undetectable, but in acromegaly typically each value is measurable, often with a fixed rate of secretion (201).
Oral glucose tolerance test: Failure of normal suppression of serum growth hormone following administration of oral glucose remains the ‘gold-standard’ biochemical test (202). 75 g of oral glucose is given at 9 am to the fasting patient and plasma glucose and serum growth hormone levels are measured at baseline, 30, 60, 90, 120 (and 150) minutes thereafter. In normal subjects, growth hormone levels suppress to undetectable values (typically <0.1 ng/ml) when ultrasensitive assays are used, whilst in acromegaly serum growth hormone remains detectable, and in approximately 30% of cases there is a paradoxical increase (199). In conventional practice failure to suppress serum growth hormone to a level < 0.4 ng/ml following ingestion of glucose supports the diagnosis of acromegaly. The use of this test also detects those patients with impaired glucose tolerance or diabetes mellitus other than individuals with poor diabetes control (203). False-positive results can be seen in conditions where GH levels are elevated such as stress, type 1 diabetes mellitus, cirrhosis, chronic renal failure, during adolescence, and by drug use (L -dopa, heroin, estrogen 201,204).
TRH test: In cases of remaining doubt about the diagnosis of acromegaly, a TRH test can be used (200 mg of thyrotrophin releasing hormone given intravenously with serum measurement at 0, 20 and 60 minutes). In normal subjects TRH inhibits growth hormone secretion with a fall in serum concentration, whilst approximately 60% of patients with acromegaly demonstrate a paradoxical rise in growth hormone levels (205). In mild disease with relatively low GH levels, TRH stimulation has been suggested to confirm early diagnosis (206).
Others: IGFBP3 levels correlate with mean 24 hour GH levels and IGF-1 levels, but due to the wide overlap with normal and diseased individuals, is a poor parameter for measuring disease activity (207). In the rare patient in whom a non-pituitary etiology is suspected, measurement of serum GHRH may be performed, typically with very elevated levels occurring in ectopic GHRH syndromes such as neuroendocrine tumors. Basal serum prolactin should also be measured as prolactin may be co-secreted with growth hormone in up to a third of patients with acromegaly, which often indicates therapeutic responsiveness to the use of dopamine agonists. In those with hyperprolactinemia the presence of macroprolactin should be excluded (208).

In patients with atypical clinical symptoms such as GI symptoms, hypoglycemia, renal stones, clinicians should consider exploring possibility of coexisting genetic tumors or extra pituitary source of GH abnormality, such as carcinoid tumors.

Biochemical Tests Used to Define Disease Activity to Guide Outcomes

Octreotide test: Acute challenge with octreotide 100mcg sc dose can help predict response to SSA when hourly values are measured over 6 hours (209,210). Not all clinicians perform this test and many treat with a long-acting SSA independent of a challenge test (211).
IGF-1 levels: IGF-1 levels can take over three months to normalize in the post-operative period and until then cannot be reliably used to guide management. In longer term the aim of treatment is to keep levels within the normal ranges (52). In patients with Pegvisomant therapy, IGF-1 is the variable that guides response to treatment (212).
Random GH levels: Random levels can be used to define surgical cure and values as early as day 1 can be undertaken if no pre-operative GH suppressing therapy was used, bearing in mind the effect of post-operative stress. A serum GH <0.4 µg/L favors disease remission and a level <1 µg/L indicates good control and normalization of the mortality risk (151).
OGTT: OGTT can be undertaken to evaluate post-operative outcome if random GH values >1µg/L. A nadir GH cut off of <1µg/L is associated with better long-term outcomes and would be considered as good control. Endocrine Society guidelines suggest GH nadir of less than 0.4mcg/ L could be used to define disease remission (151) It has been suggested that post-operative OGTT at 3 months is more appropriate than an early test in 3 weeks and is likely to help avoid false positives (213).
Other tests: The GH day curve test is rarely required for long term monitoring. The mean GH value of <2.5µg/L correlates better with disease control but it is unreliable in patients who have undergone radiotherapy due to alterations of GH pulsatility pattern (214). Post-operative TRH test has been suggested to predict long term disease remission, but again is rarely required (211).

Radiological Assessment

Historically the diagnosis of pituitary tumors causing acromegaly was made on the basis of changes to skull bones with demonstration of enlargement of the fossa. With advances in radiology, pituitary MRI with gadolinium enhancement is considered the optimal modality and should be undertaken to determine size of the tumor, define tumor characteristics and assess threat to surrounding structures. At diagnosis, more than 70% of patients with acromegaly have a macroadenoma (≥10 mm in diameter) which often extends laterally to the cavernous sinus or superiorly to the supra-sellar region. Younger patients often present with more aggressive disease, with more invasive tumors which often extend inferiorly. On T1 weighted images the pituitary adenoma tends to be of lower signal intensity than the surrounding normal gland and enhances less briskly than the normal gland after injection with intravenous gadolinium contrast. Tumors >15mm, supra-sellar extension, and cavernous sinus extension of the tumor has been associated with lower rate of surgical cure (215). Radiologic grading of pituitary tumors using KNOSP classification is widely used in predict disease invasiveness and tumor response. Some studies report cavernous sinus invasion as the strongest factor predicting surgical outcome (216,217).

In the last decade there is substantial evidence to suggest that hypointensity on T2 weighted MRI is suggestive of good response to somatostatin analogue SSA therapy (218,219). Based on histological characteristics, hypointense T2 weighted tumors correlate with a densely granulated histological subtype and tend to be less aggressive in behavior (220,221). In the post-operative phase, MRI should be considered approximately 3 months after surgery to allow post-operative changes to settle. Subsequent surveillance scans should be guided by biochemical markers as they guide correlation with tumor recurrence. Unless concerned, unenhanced imaging may be preferred for patients expected to require long term surveillance to reduce exposure to gadolinium (222). There are limitations of MRI when it comes to evaluation of persistent or residual disease. In these cases, use of C11 Methionine Positron Emission technology MET-PET with co-registration of volumetric MRI has been found to provide valuable information to guide repeat surgery or targeted gamma knife surgery (223,224). In patients with previously reported empty sella or residual parasellar tissue where surgeons would struggle to consider intervention, the information provided by MET-PET has been found to be useful with positive outcomes. Other tracers that have been studied and reported to guide management include N13 ammonia PET, DOTATATE PET (225,226).

In rare cases of ectopic GHRH secretion, a pituitary MRI can sometimes help differentiate between a pituitary adenoma and hyperplastic pituitary tissue (53). The use of somatostatin receptor scintigraphy (particularly Gallium Dotatate) is useful to correlate the source of ectopic hormone production to an unexpected finding on a conventional body imaging.

Figure 6. Enlargement of pituitary fossae on lateral skull x-ray.

Figure 7. MRI demonstrating a somatotroph macroadenoma of the pituitary gland.

Neuro-Ophthalmological Testing

Neuro-ophthalmological assessment should be undertaken in all patients with macroadenomas, especially where tumor is visibly contacting the optic chiasm. At the initial consultation visual acuity should be assessed with the use of Snellen charts and fundoscopy performed to exclude optic atrophy, retinal vein engorgement, or papilledema from pressure on the visual pathways. Visual fields may be assessed by confrontation using a red pin. Patients with any clinical symptoms or evidence of optic chiasmal compression from imaging studies require formal assessment of visual fields with formal perimetry or visual evoked responses, stimulating each half field in turn. Optical coherence tomography should be used to assess retinal nerve fiber layer as a marker of chiasmal damage.

Although permanent loss of vision and/or visual field defects usually result from long standing optic chiasmal compression, the shorter the time of compression the easier and more complete is the reversal of any visual field deficit. Surgical decompression may result in rapid improvement in visual fields within hours or days, although the presence of optic atrophy reduces the likelihood of this occurring. Because onset is often insidious, patients may be unaware of any alteration in their vision, although once documented its presence requires them to inform the vehicle licensing authority as driving ability may be impaired. An exception to this usual gradual deterioration is pituitary hemorrhage when visual loss may be sudden with a loss of central vision and development of bitemporal field defects and possible ophthalmoplegia often accompanied by changes in higher mental function.

Assessment of Pituitary Function

Assessment of the integrity of the other pituitary hormones needs to be performed by a combination of the appropriate basal and dynamic tests. These are mentioned in other Endotext chapters. Prior to and following pituitary surgery, both residual pituitary function and the growth hormone secretory status should be evaluated. Basal endocrine testing for early morning cortisol, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, testosterone/estrogen, prolactin, and serum & urinary osmolality, should be performed. Where there is doubt, a provocative test should be made of ACTH reserve.

Histological Assessment

Histology provides valuable information to determine tumor characteristics. Keratin staining is essential to distinguish between densely granulated and sparsely granulated tumors. Further use of immunohistochemical IHC staining modalities is increasing found to help understand tumor behavior. Details of various histological subtypes are highlighted in table 2. IHC staining for different transcription factors Pit-1, SF-1, or Tpit along with hormonal staining is used to understand the tissue of origin. Ki67 labelling index is widely used to determine proliferation rates, though it does not always predict tumor behavior. Evaluation of somatostatin receptor SSTR expression is used to guide response to SSA therapy. Additional immunostaining modalities used in recognition of familial tumors are menin, p27, AIP and SDH expression (227).

Genetic Testing

Over the last decade, we have seen an increasing understanding of the role of specific genes and mechanisms responsible for development of pituitary tumors. The genetic disorders associated with acromegaly are summarized in table 4.

The most exciting developments relate to recognition that aryl hydrocarbon receptor interacting protein (AIP) mutations can account for familial pituitary adenomas. Such cases may present in younger patients with more aggressive tumors, including somatotroph adenomas. Testing for AIP mutations (FIPA) should be considered for any patient with a family history of pituitary tumor, especially GH and/or prolactin secreting tumors (228). This is particularly so, in those presenting at younger age (<30 years) with aggressive pituitary adenoma. There is some evidence that the response to somatostatin analogue treatment is reduced in acromegaly associated with AIP mutation (229). Data from the German Pituitary Registry suggest that even in younger patients with acromegaly that the prevalence of AIP mutations is low (<5%) (230). X-LAG X-linked acrogigantism mutation is reported to lead to about 80% cases of pre-pubertal acromegaly (231). GH secreting adenoma is reported to occur in approximately 6% of patients with MEN1 (232). In practice patients presenting at a young age with acromegaly, co-existent hypercalcemia, and certainly those with a family history of pituitary tumors should be considered for MEN1 and AIP gene sequencing, respectively.

Table 4. Genetic Disorders Associated with Acromegaly (233,234)
Genetic condition	Gene implicated	Inheritance	Clinical features
X-LAG	GPR101	X-linked dominant or sporadic	Mostly females Age <5 years Hyperprolactinemia at diagnosis Can be de-novo and family history may be absent
Familial isolated pituitary adenoma	AIP	Autosomal dominant or sporadic	Younger males Higher GH levels Poor response to SSA therapy
MEN1	MEN1	Autosomal dominant or sporadic	Presence of primary hyperparathyroidism, pancreatic disease along with pituitary adenoma
MEN4	CDKN1B	Autosomal dominant	Presence of pituitary and parathyroid neoplasms with pheochromocytomas, thyroid and other tumors
Carney complex	PRKAR1A	Autosomal dominant	Skin pigmentation, Atrial myxoma, GH or PRL excess, Cushing’s due to primary pigmented nodular adrenocortical disease Somatrotroph hyperplasia or multifocal adenoma
Mc Cune Albright	GNAS	Autosomal dominant or sporadic	Average age of diagnosis 23 years Acromegaly seen in 20-30% cases Hyperprolactinemia present at diagnosis Café-au-lait spots Peripheral Precocious puberty Somatrotroph hyperplasia
SDH mutation	SDHx	Autosomal dominant or sporadic	Phaeochromocytoma, paraganglioma and primary hyperparathyroidism present
Neurofibromatosis	NF1	Autosomal dominant	Few case reports of children and adolescents with optic glioma, rarely adenoma reported Mechanisms unclear

Investigations for Complication Screening

Acromegaly is associated with a significant number of co-morbidities most of which are present at time of diagnosis. Once the diagnosis is established it is essential to evaluate for complications such as DM, hypertension, dyslipidemia, osteoporosis, sleep apnea, carpal tunnel syndrome, quality of life, and neoplasia. A suggested screening approach is outlined in table 5.

Table 5. Suggested Strategy for Screening for Complications (52,145,222)
Co-morbidity to evaluate	Screening test	Frequency
Hypertension	Measurement of BP Ambulatory BP monitoring in selected cases	6 monthly
DM	Hba1c, FBG OGTT in selected cases	At diagnosis and 6 monthly if normal
OSA	Clinical evaluation and Epworth scale Polysomnography for confirmation	At diagnosis and annually
Cardiomyopathy	ECG Echocardiography	At diagnosis and 3-5 yearly if normal
Dyslipidemia	Lipid profile	At diagnosis and 6 monthly
Colon polyps	Colonoscopy	At diagnosis in patients >40 years age, and 10 yearly if normal, 3-5 yearly if polyp noted and IGF-1 elevated
Thyroid nodules	Clinical evaluation Thyroid US guided by examination	Annually
Vertebral disease	Bone morphometric study using thoracic x-ray, thoracic and lumbar spine x-ray DEXA	At Diagnosis and yearly, guided by symptoms
Cerebral aneurysm	Cerebral MR angiography	Infrequently
QOL	ACROQOL	Annually

DIFFERENTIAL DIAGNOSIS

Patients with tall stature are frequently suspected to have acromegaly, but absence of soft tissue changes and normal biochemistry should prompt investigations for alternative causes such as Marfans syndrome, homocystinuria, or a familial trait. Pseudoacromegaly or acromegaly mimics are rare disorders with clinical manifestations strongly suggestive of GH excess, but without any biochemical evidence favoring the diagnosis. Pachydermoperiostosis is reported more commonly in males and has an autosomal recessive inheritance. It has been reported secondary to HPGD mutations or SLCO2A1 mutation, which lead to an increase in prostaglandin E2 (235).

MANAGEMENT OF ACROMEGALY

Given the chronic nature and associated significant increased morbidity and mortality of acromegaly, treatment is required for almost all patients. Three modalities of treatment are available: surgery, pituitary irradiation, and medical therapy. All of these have advantages and disadvantages and more than one modality is frequently needed, sometimes all three. The decision as to whether to treat and the modality employed must be based on a number of factors, including patient age and general health, wish for fertility, severity of disease and any associated complications, and the risk/benefit ratio of the proposed treatment modality. The goals of treatment are summarized in Table 6.

Consensus guidelines define goals for treatment of acromegaly. These include achieving an age-matched normal range IGF-1 and GH <0.4 mcg/L (236).

Table 6. Acromegaly- Aims of Treatment

1. Removal of the pituitary tumor and resolution of mass effects

2. Relief of the symptoms and signs of acromegaly

3. Restoration of normal rates of secretion of growth hormone and IGF-1

4. Maintenance of normal anterior pituitary function

5. Prevention of recurrence

6. Assessment and treatment of chronic complications

Whilst the general principles of these aims are accepted by all endocrinologists, there remains considerable controversy as to the degree of growth hormone reduction that should be the target and what level should be regarded as normal. The use of sensitive growth hormone assays has demonstrated that abnormal patterns of growth hormone secretion can remain despite reduction in mean circulating concentrations to extremely low levels, and thus complete restoration to normality is often not achieved. Early epidemiological reviews, particularly those documenting the results of surgery, tended to regard a mean level of less than 5 ng/ml as being satisfactory. It has become clear in recent years that the excess mortality associated with acromegaly can be significantly reduced and indeed restored to that of the normal population by aggressive treatment and reduction of serum growth hormone concentrations to a mean level of less than 1 ng/ml and/or a serum IGF-1 within the aged-matched reference range. Thus, rather than using the word cure, it is may be more appropriate to consider an average growth hormone concentration of ≤ 1 ng/ml as representing a "safe" level, whereas current consensus is to aim for <0.4 ng/ml (236).

Surgery for Acromegaly

Trans-sphenoidal surgery is the initial treatment of choice for most patients. Originally performed by Harvey Cushing in 1910, the lack of adequate visualization prevented its reintroduction for routine use until the mid-1970's. With modern equipment and in experienced hands, it is a safe procedure with a low complication rate and mortality of less than 0.5%. The most commonly used approach is with the patient in a semi-reclining position via a mid-line nasal route. Using a sub-labial or direct nasal approach, the mucosa is cleaved off the nasal septum providing access to the sphenoid sinus and subsequent removal of the fossa floor. A less satisfactory alternative approach is via the ethmoidal sinus. Pituitary adenomas are usually soft and easily removed with curettes although firmer and larger tumors may require piecemeal removal. Using this technique, even tumors with a significant suprasellar extension can be removed via the trans-sphenoidal route, although massive tumors may require a craniotomy. Such transcranial surgery is however associated with increased morbidity and mortality and is rarely required. More recent surgical techniques include the use of intra-operative MRI (237) and intra-operative growth hormone measurement (238). The development of endoscopic trans-sphenoidal surgery offers several advantages over the conventional technique, and is the now the method of choice. Reported advantages over the microscopic technique include superior tumor clearance, especially suprasellar extension, less surgical morbidity, fewer complications, and reduced post-operative discomfort (239), though evidence suggests endoscopic surgery and microsurgery yield similar outcomes in the most experienced hands (240). Endonasal endoscopic surgery, with ability to resect cavernous sinus located adenoma will increasingly be standard of care in pituitary surgery.

A key recent development in the management of acromegaly internationally is the formalization of a team approach, with endocrinologist, neurosurgeon, specialist nurses, oncologists, radiologists, and histopathologists increasingly working as a single-team, making consensus decisions in a timely and coordinated fashion. There is general acknowledgement that functioning pituitary tumors are best managed in centers with larger volume and experience of rarer conditions (236). Certainly, this practice is increasingly the case in Europe and the USA.

The success rate of trans-sphenoidal surgery depends on several factors: (i) the size of the tumor, (ii) pre-operative growth hormone values and (iii) the skill and experience of the surgeon and (iv) most importantly cavernous sinus invasion. A predictive model using age, KNOSP classification, and pre-operative GH levels has been proposed to predict surgical remission and guide pre-operative medical management and long term management (241). Although different series have often used different criteria to determine success rates, in experienced hands post-operative mean growth hormone levels of less than 1 ng/ml should be achieved in 70%-90% of microadenomas and 30%-50% of macroadenomas (52). Pre-treatment of patients with somatostatin analogues before trans-sphenoidal surgery is increasingly becoming standard practice, even if early surgery is being planned, as it results in significant shrinkage (approximately 50%) of the adenoma and may improve the subsequent surgical cure rates (242).

Complications of trans-sphenoidal surgery include diabetes insipidus, CSF rhinorrhea, meningitis, and hypopituitarism. Diabetes Insipidus is usually transient but may be permanent in approximately 5% of cases depending on the criteria for its diagnosis. A serum osmolality of greater than 295 mosmols/l with a simultaneous urine osmolality of less than 150 mosm/l is confirmatory. It responds well to desmopressin (DDAVP, subcutaneous, oral, or intranasal).

Radiotherapy in Acromegaly

Radiotherapy in the management of pituitary disorders including acromegaly is discussed in detail in another Endotext chapter. Pituitary irradiation is usually used as an adjunct to pituitary surgery when growth hormone levels remain elevated. In elderly patients or those unfit for surgery, it may rarely be used as first-line therapy (243). There are several techniques that have been used: conventional mega-voltage external irradiation, stereotactic single high dose irradiation, interstitial implantation of yttrium ⁹⁰ seeds, and whole particle proton beam therapy. Only the first two will be discussed here.

Conventional mega-voltage irradiation has been in routine use for over 40 years and consequently there is a wealth of experience principally relating to it being both a safe and effective technique. A linear accelerator is used as the source; less satisfactory is a cobalt source. Irradiation is focused onto the pituitary fossa using modern CT/MRI imaging and planning, which allows for accurate dosimetry and minimal variation in the daily dosage to surrounding structures, using IMRT. This is particularly so for the optic chiasm, damage to which is avoided by the use of daily fractions of less than 200 cGy. The majority of centers advocate a total dose of 4500 cGy given in 25 fractions of 180 cGy over 5-6 weeks via a minimum of three fields (one frontal and two temporal). Numerous studies have confirmed the efficacy of such mega-voltage irradiation with a 50% fall in growth hormone values occurring in the first two years, regardless of basal levels, followed by a continuing exponential decline thereafter (244). The majority of patients therefore do eventually achieve a level of less than 2 ng/ml, although the interval to reach this depends on the baseline levels. A similar response is seen with IGF-1 with approximately 60% of patients eventually achieving a normal serum level after 10 years. Although it is recognized that pituitary irradiation is associated with several potential adverse consequences, these are rare when irradiation is delivered properly, other than an increased prevalence of hypopituitarism. At ten years after irradiation, approximately 60% of patients are hypogonadal, 50% ACTH deficient, and 40% requiring thyroxine replacement. However, the prevalence prior to irradiation, either due to the pituitary tumor itself or previous surgery should be taken into account, with baseline figures being 40% hypogonadal, 35% ACTH, and 15% TSH deficient (244). Other concerns include development of secondary tumors in up to 2% of cases, radiation induced optic neuropathy in up to 5% cases, cerebrovascular events in up to 20% of cases over 20 years duration, brain necrosis, and psychocognitive impairment (245-247).

Stereotactic single high dose pituitary irradiation using either the gamma knife (radiosurgery) or stereotactic multiple arc radiotherapy (SMART) has received increasing attention in recent years as an alternative to conventional irradiation. These techniques permit the delivery of a single high dose of irradiation to a previously mapped area whilst also ensuring a rapid reduction in radiation exposure to surrounding structures. Median dose delivered is 15-35Gy and invariably achieves good tumor control at 5 years follow up and about 50% biochemical remission at 5 years (247)Care needs to be taken with tumors close to the optic chiasm. Initial impressions suggest that growth hormone levels fall to normal earlier than after conventional radiotherapy, but that hypopituitarism occurs just as often (248). Side effects of secondary brain tumors and cerebral vasculopathy seem to be lower but long term studies are awaited (247). Although the stereotactic technique has clear advantages over conventional external irradiation in terms of precise mapping to a specified tumor volume, it may not encompass tumor tissue that is not visualized radiologically. This is in contrast to conventional irradiation which is usually configured to encompass the whole of the pre-operative tumor volume, and thus will treat tumor beyond the resolution of imaging techniques. It is for this reason that stereotactic irradiation should be seen as complementary to conventional irradiation.

Medical Therapy of Acromegaly

Three different types of medical therapy are currently used in the treatment of acromegaly, dopamine agonists, somatostatin analogs and growth hormone antagonists.

DOPAMINE AGONISTS IN THE TREATMENT OF ACROMEGALY

From their discovery and synthesis in 1971 until the introduction of somatostatin analogs in the mid-1980’s, dopamine agonists, such as bromocriptine, were the sole medical therapy for acromegaly. However, they are relatively ineffective and whilst approximately 80% of patients will show a reduction in growth hormone levels, only about 10-15% achieved a mean level of less than 2 ng/ml (249). Furthermore, the doses required, often 20 - 30 mg of bromocriptine per day, are much higher than those needed for prolactin-secreting pituitary adenoma. Consequently, the side effects of nausea, headache, dizziness, postural hypotension, and nasal stuffiness tend to be worse, although can be minimized by taking the drug in the middle of a main meal to slow absorption and most patients will demonstrate tachyphylaxis. Unlike in patients with prolactinomas (where an excellent treatment response is expected), there may be only a modest reduction in tumor size but this is usually insignificant. Cessation of treatment results in rebound growth hormone hypersecretion. The use of bromocriptine in acromegaly is limited. The development of the long-acting dopamine agonists such as cabergoline offered greater convenience and reduced side effects, although again high doses of up to 4 mg per day may be needed (250). A meta-analysis has demonstrated that its use can achieve normalization of IGF-1 levels in 34% of patients (251). There are no accurate predictive tests as to which patients will respond to dopamine agonists, but it has a place in the management of mixed growth hormone and prolactin secreting tumors and patients with mild IGF-1 elevation. Combination therapy with SSA is frequently used in patients with co-secreting tumors with good effect (252).

Fig 8. Plasma IGF-1 and GH responses to dopamine agonist suppressive therapy in patients with pure GH secreting tumors. The upper squares indicate the pretreatment levels and lower squares correspond to the concentrations obtained by progressively increasing the weekly dose of cabergoline i.e., 1.0, 1.75, and 3.5 mg, respectively. Note the log scale for GH.

SOMATOSTATIN ANALOG TREATMENT OF ACROMEGALY

The development of octreotide (Sandostatin, Novartis, Basel, Switzerland) a synthetic somatostatin analog, represented a major advance in the treatment of acromegaly. In contrast to the short half-life of native somatostatin (approximately 90-seconds), the 8 amino acid octreotide has a half-life of about two hours. Following a single 100 mcg dose, there is prolonged suppression of growth hormone which lasts for several hours, and indeed this response to a single dose can be used to predict the long-term efficacy of octreotide. It is administered by subcutaneous injection and thus a thrice-daily regimen results in stable drug concentrations and maximal effect. More than 90% of patients show a reduction in growth hormone levels, with approximately 50-60% achieving levels of less than 2 ng/ml and a normal serum IGF-1 level. The usual doses are between 100-200 mg three times daily although occasional patients may require higher doses. This biochemical improvement is matched by rapid clinical improvement. The efficiency of octreotide and other somatostatin analogs (SSAs) such as lanreotide is linked to their preferential binding of the human somatostatin receptor type 2 (SSTR2) with reduced or absent binding of SSTR1, SSTR3, SSTR4 or SSTR5. Somatostatin analogs also have additional and independent, but poorly understood, analgesic properties on the headache associated with acromegaly.

Since the introduction of short-acting octreotide, depot formulations of somatostatin analogs have become available. These consist of the active drug incorporated with microspheres of biodegradable polylactide and polyglycolide polymers which allow the slow release of analog after intramuscular injection. There are currently three such preparations available, octreotide LAR (Sandostatin LAR, Novartis) which is given at a variable dose of 10 mg, 20 mg or 30 mg at recommended four weekly intervals, lanreotide (Somatuline Autogel, Ipsen Biotech, Paris, France), which is given as a single dose of 60-120 mg every 28 days as a sub-cutaneous depot formulation, and the more recently licensed Pasireotide LAR. Pasireotide has increased affinity for SSTR5, and this has led to a license for the treatment of Cushing’s disease in addition to acromegaly. The SSA medications are also used in the treatment of neuroendocrine tumors arising outside the pituitary gland, in particular small bowel carcinoid tumors and pancreatic neuroendocrine tumors. Sandostatin and Lanreotide Autogel are of similar efficacy in suppression of growth hormone and IGF-1 with safe growth hormone levels (<2 ng/ml) occurring in approximately 60-70% of patients (253), although a meta-analysis of patients unselected for somatostatin responsiveness indicated that normalized IGF-1 levels and safe growth hormone levels occurred in a higher proportion of LAR treated than lanreotide treated patients (254).

Regardless, of the comparative effects, there is variability in individual patient’s sensitivity to these analogs and more than 90% of patients who achieve adequate control with 4 weekly octreotide LAR injections will also do so with 6 weekly injections (255). Consequently, careful dose titration needs to be performed on each patient. This is particularly important given the cost of these depot formulations; in the UK, the approximate annual cost of octreotide LAR given 4-weekly is £8000 for 10 mg injections, £11000 for 20 mg and £14000 for 30 mg, whilst the cost for lanreotide Autogel 90 mg is approximately £10000 per annum. Biosimilar agents will increasingly become available perhaps with reduced cost.

Pasireotide is a novel cyclohexapeptide somatostatin analogue which is selective for SSTR2, 3 and 5, but also shows increased binding to SSTR1 compared to octreotide (256). The extended receptor affinity of pasireotide has led to it being referred to as a “second generation somatostatin analogue” with the original depot formulations being termed “first generation” analogues. More recent clinical trial data relating to Pasireotide in acromegaly indicates that this agent is modestly more potent than Sandostatin LAR in achieving control of GH and IGF-1, has long term safety, and also has a place in the management of seemingly octreotide resistant disease (257). A Phase III study showed that in new presentations of acromegaly achievement of control of GH and IGF-1 is superior with pasreotide (over octreotide) with about 20% patients achieving complete remission at 6 months, in a group resistant to first generation SSA therapy, suggesting that pasireotide may replace the earlier SSAs in treatment strategies in the future (258). Recent data from the PAOLA extension study showed that of the patients who achieved biochemical control at some point, 65.6% cases did so after 6 months of treatment. Increasing dose from 40-60mg allowed better remission rates (additional 28%) with reasonable safety profile (257). The drug seems to be particularly useful in the management of severe headaches in patient with acromegaly.

Oral octreotide as an agent coupled to a transient gut absorption enhancer and has been very recently approved by the FDA. Phase 3 studies have shown that it helps achieve about 65% control of IGF-1 and GH when switched from injectable SSA and sustains the benefit in about 90% individuals for at least 13 months of follow up (259). It is prescribed in the dose of 40-80mg per day and studies have demonstrated better absorption in a fasting state (260). The most commonly reported adverse effects include headache, nausea, and arthralgia.

The side effects of somatostatin analogs are related to the widespread distribution of somatostatin and include effects on the gastrointestinal system, comprising colic type abdominal pain, diarrhea, flatulence, and nausea, although these tend to resolve with time. In the long-term gastritis occurs in a significant proportion of patients and perhaps most significantly gallstones form in approximately 50% of patients after two years of use. This is due to both an inhibition of gall bladder contraction and alterations in the composition of bile with cholesterol supersaturation. However, perhaps due to the gall bladder paresis, the majority of these remain asymptomatic. The effects of SSAs on glucose metabolism are multifactorial. While they improve insulin sensitivity by reducing growth hormone levels, they also exert direct inhibitory actions on insulin secretion by the pancreatic cells. The net result is normal glucose tolerance in the majority of patients. With their improved patient convenience, there have been suggestions that these depot formulations should be used as first-line treatment for acromegaly. However, their increased cost and the need for continuing treatment should be borne in mind. At present, there remains general consensus that whilst they may have a role prior to surgery to try and decrease tumor size, their major place is post-operatively as an adjunct to irradiation whilst waiting for growth hormone levels to fall. Provisional evidence suggests that treatment of acromegaly with somatostatin analogs prior to surgery improves the cardiovascular risk and respiratory status and may therefore have a place in larger and invasive tumors (261,262). Patients who remain uncontrolled despite the use of these somatostatin analogs may gain additional benefit with the addition of a dopamine agonist, but this is the exception rather than the rule. The incidence of hyperglycemia or diabetes is higher when patients are treated with pasireotide. This and the cost of the drug have thus far limited its use in the UK, though other health care economies have more readily incorporated pasireotide into the acromegaly treatment algorithm.

GROWTH HORMONE ANTAGONISTS IN ACROMEGALY

The development of Pegvisomant, the novel growth hormone receptor antagonist, is a major advance in the treatment of acromegaly. The development of this molecule utilizes the knowledge that the growth hormone molecule contains two distinct sites which bind to two corresponding unique sites on the respective growth hormone receptor dimer. Pegvisomant is a modified recombinant growth hormone molecule which has increased affinity to the first growth hormone receptor binding site but with decreased affinity to the second binding site. Thus, receptor dimerization and subsequent signal transduction is prevented. Its conjugation with polyethylene glycol (PEG) increases its molecular size, prolongs its half-life and reduces its antigenicity. Based on long term experience, some authors propose Pegvisomant to be most effective medical therapy to date and suitable as first line intervention in selected cases (263). In a study of 152 patients treated for up to 18 months, normalization of IGF-1 occurred in 90% of patients, although doses of up to 40 mg a day were required (264). Growth hormone levels cannot be measured in routine assays as the drug itself interferes with growth hormone assays and pituitary-derived growth hormone increases modestly. Pegvisomant is currently administered as a daily subcutaneous injection of approximately 1 ml in volume. Theoretical concerns exist regarding the increase in circulating growth hormone levels due to the loss of any negative feedback effects on the tumor, but although experience is still limited there is no evidence to date of risk of pituitary tumor growth (263).

Pegvisomant is generally well tolerated although abnormalities of liver function occur in some patients. Its major use is for patients who are resistant to SSAs, either as a sole agent or as an additive agent. A study observed that the combination of 4-weekly octreotide LAR and weekly Pegvisomant normalized IGF-1 in more than 90% of patients with active disease who were not controlled with octreotide alone (265). Other suggestions for its use have been in patients with diabetes or impaired glucose tolerance, in whom SSAs might worsen glycemic control. Higher doses may be required in patients with severe disease, DM, and obesity. However, the change in dosing frequency and additional cost needs to be weighed against the use, if required, of simple oral hypoglycemic agents. The major drawback of Pegvisomant other than its usual requirement for daily injection, as opposed to the 4-6 weekly administration of SSAs, is its cost of approximately £3000 per per month, which can amount to £36000 per annum for patients resistant to SSAs (266).

A combination study demonstrated improved IGF-1 control with Pegvisomant and cabergoline, an approach which might enable a lower dose of the Pegvisomant to be used with reduced costs (267). Emerging data suggest that Pegvisomant may be an effective long-term treatment for acromegaly (263,268). Several European countries have registries providing regular outcome data related to Pegvisomant treatment in acromegaly. However, cost and approval restrictions mean that Pegvisomant is not yet universally available. Combination treated with SSA and Pegvisomant is likely the most effective medical strategy. Table 7 contains a summary of reported studies assessing effectiveness and safety of this treatment approach.

PEG pegvisomant, SRL long-acting somatostatin receptor ligand, QoL Quality of life, OL open-label, LAN Lanreotide, OCT Octreotide LAR, OGTT 75 g oral glucose tolerance test, NA not available

^aInclusion criteria: responders to daily PEG monotherapy (presumed previously uncontrolled on SRL therapy), or partial responders to the highest marketed doses of either PEG at 3 months or SRL at 6 months

^bPost hoc analysis: eight patients whose mean IGF-1 levels were similar while on pegvisomant monotherapy and during the co-administration period were able to reduce their weekly pegvisomant dose by 50 %

^cDefined as > 2 × ULN in this study

^dDifferent study criteria for IGF-1 normalization: defined by either end-of–study IGF-1, or lowest IGF-1 achieved

^ePrevious pituitary surgery: 1/4; Primary medical therapy: 3/4; none had radiotherapy

^fPrevious pituitary surgery: 2/14; Primary medical therapy: 11/14; one patient had radiotherapy

^g12/21 patients who did not achieve normal IGF-1 received PEG < 20 mg/day

^h2/3 patients with elevations >10 × ULN received OCT 60 mg/28 days

ⁱNone had radiotherapy

Table 8. Medical Agents for Acromegaly Including Drugs Under Trial (Most Relevant Targets in Bold). (269-272)
Agent	Route of administration	Molecule	Target	Dose	Side effects
Cabergoline	Oral	Dopamine agonist	DR2	1-4mg /day	Nausea, headache, dizziness, postural hypotension, and nasal stuffiness. Rare concerns of mood disorders and valvular fibrosis
Octreotide LAR	IM	Somatostatin analog	SSTR2 - SSTR5	10-40mg 4 weekly	Gastrointestinal side effects, GB sludge, reduced GB contractility, cholelithiasis, hypothyroidism. Variable effect on glucose. Rare sinus bradycardia, alopecia
Lanreotide ATG	Deep SC	Somatostatin analog	SSTR2 - SSTR5	60-120mg 4 weekly
Pasireotide LAR	IM	Somatostatin analog	SSTR1, SSTR2, SSTR3, SSTR5	40-60mg 4 weekly	Same as above. Also, Hyperglycemia
Pegvisomant	SC	GH receptor antagonist	GH receptor	10-40mg / day	Injection site reactions, abnormal liver enzymes, increase tumor size?
Tamoxifen	Oral	Selective estrogen receptor modulator		20-40mg / day	Bone marrow suppression, gynecologic malignancies, hepatotoxicity, ocular effects, thromboembolic events (271)
Octreolin®	Oral	Somatostatin analog	SSTR2 - SSTR5	40-80mg/day	Nausea, bloating, diarrhea, GB stones, dysglycemia
THERAPIES UNDER TRIAL
Glide Octreotide Acetate (GP02)	Needle-free version of regular octreotide acetate	Somatostatin analog	SSTR2 - SSTR5	Immediate release drug, details unclear	Trial data not available
IF-2984®	SC	Somatostatin analog	SSTR1, SSTR2, SSTR3, SSTR5	Immediate release drug, details unclear	Trial data not available
CAM2029	SC	Somatostatin analog		20mg monthly depot	Similar to Other SSA
DG3173 (Somatoprim, now called as Veldoreotide)	SC	Somatostatin analog	SSTR2, SSTR4, SSTR5	100-1800µG TDS	Injection site reactions, GI side effects
ATL1103	SC	Antisense molecule	GH receptor (mRNA)	200mg twice weekly	Injection site reaction
Q-chip Octreotide	SC	Somatostatin analog	SSTR2 - SSTR5	10-30mg weekly	Diarrhea, DM
Botulinum neurotoxin SXN101959	–	Engineered neurotoxin	GHRH receptor	1mg/kg
Intravail Octreotide ProTek ®	Oral/nasal	Somatostatin analog	SSTR2 - SSTR5
VP-003 hydrogel formulation	SC implant	Somatostatin analog		84mg 6 monthly	Similar to SSA

PERSONALISED MANAGEMENT OF THE PATIENT WITH ACROMEGALY

Acromegaly is a rare condition and is best managed by expert teams with a personalized approach. Patient factors, the presence of co-morbidity, quality of life, and resource availability each influence the approach to management. The Endocrine Society guidance is commonly considered and increasingly biomarkers of disease status and activity are used to guide decision making.

Surgery with an intention of cure, or debulking remains the first line of management in most cases. Current evidence suggests that in cases of growth hormone secreting microadenoma, surgery alone will result in achievement of ‘safe’ growth hormone levels in approximately 70-90% of patients. As per the Knosp criteria tumors that cross the lateral tangent of the intracavernous and supracavernous internal carotid arteries are classified as grades 3A, 3B or 4 and are considered invasive (273,274). Surgical remission rate falls when an invasive macroadenoma (<50%) or a giant adenoma (<20%) is present, in contrast to non-invasive tumors (76%). Those with the highest pre-operative growth hormone concentrations, large tumors, or ones with invasion of cavernous sinus are least likely to be ‘cured’ by surgery alone (275). Older age and lower GH levels are associated with likelihood of cure (276). There may circumstances where consideration could be given for the use of cabergoline for IGF-1 values below twice the upper limit of normal. Use of SSA could be considered pre-operatively in patients with an intention to reduce disease burden or tumor volume prior to surgery to facilitate intervention.

Several biomarkers have been suggested to help guide response to first generation SSA. In a pre-operative patient T2 hypointense lesion is more likely to respond to SSA. Most of the other biomarkers are guided by histology outcome such as presence of densely granulated tumor, anti-Cam5.2 staining pattern, SSTR2a expression, and Ki-67 are well-established IHC biomarkers for response to first-generation SSA therapy (277). SSTR2a expression is also as useful marker for Pasireotide response (278). Other markers suggested to predict poor SSA response include low AIP expression, low zinc finger protein ZAC1 (a zinc finger protein expression), poor response during acute octreotide test, the presence of a gsp mutation, low expression of E-cadherin, the expression of sst5 and its truncated isoform (sst5TMD4), higher expression of miR-34a, the expression of β-arrestin, and Raf kinase expression, but these are not well validated (274). While presence of AIP expression is useful to guide use of first generation SSA, it does not seem to correlate with effectiveness of use of Pasireotide (279). There are no IHC markers to guide use of Pegvisomant, but patients with lower IGF-1 respond better (280).

Figure 9. Algorithm for management of acromegaly: Colao 2019.

TREATMENT STRATEGY IN ACROMEGALY

Figure 9 summarizes the initial and subsequent strategies and options used at each stage of patient management. Practical issues including medication and treatment availability, patient factors, and surgical expertise will each have an influence on treatment. Following confirmation of the diagnosis of acromegaly surgical treatment should be considered for all patients with a confirmed somatotroph adenoma (80). Current evidence suggests that in cases of growth hormone secreting microadenoma, surgery alone will result in achievement of ‘safe’ growth hormone levels in approximately 70-90% of patients. This figure falls when a macroadenoma (<50%) or a giant adenoma (<20%) is present. Those with the highest pre-operative growth hormone concentrations are least likely to be ‘cured’ by surgery alone. In those post-operative patients with continuing growth hormone excess, further treatment is indicated, and this can be medical or radiotherapy treatment. A second surgical procedure will result in ‘safe’ growth hormone levels in only 20% of patients. Recognizing that radiotherapy does not result in an instant lowering of growth hormone levels, medical treatment is commonly required, especially in the short-term. On average, two years following external beam irradiation growth hormone levels have decreased by approximately 50% with a further fall resulting in 75% reduction at 5 years. Newer stereotactic radiotherapy techniques, when used appropriately, may affect a more rapid reduction in growth hormone levels. However, since the tumor in such cases is usually a macroadenoma, we would only use radiosurgery as “salvage therapy” in the face of poor control of tumor secretion or regrowth following conventional radiotherapy. Available adjunctive medical options include the use of dopamine agonists, somatostatin analogs (first and second generation), and Pegvisomant. Bromocriptine will normalize growth hormone levels in only 10% of patients, although this may rise to 30% with cabergoline. Octreotide and lanreotide, particularly in their depot formulations which last 4-6 weeks, will normalize mean growth hormone levels in 70-80% of patients, and are therefore highly effective, albeit expensive. Pasireotide results in more potent GH lowering, and in many countries is becoming a key part of the treatment algorithm. The growth hormone receptor antagonist, Pegvisomant, is now well established and may be used in patients resistant to these agents. Periodic assessment with IGF-1 measurement and growth hormone profile testing should be performed at regular intervals to facilitate titration of doses and determine response to radiotherapy. Following irradiation it is reasonable to assess growth hormone status after appropriate discontinuation of medical therapies at 6-monthly intervals for 2 years and thereafter yearly. In all patients with acromegaly efforts should be made to optimize lung and cardiac function and particular attention be made to the management of cardiovascular risk factors including smoking, dyslipidemia, and abnormalities of carbohydrate metabolism. ‘Extra-hepatic acromegaly’ describes the concept that elevated GH concentration results in tissue specific pathological effects despite normalization of serum levels of IGF-1 (295). It has been postulated that combined use of SSA (to lower GH) and pegvisomant (to control IGF-1) may be the most appropriate strategy for patients who fall into this category.

Treatment of Refractory Disease

Acromegaly with invasive non-responsive adenoma, with either persistent GH excess or invasive adenoma is a rare and difficult management problem often needing multi-modal therapy. Table 7 summarizes data from studies reporting use of combination medical therapy in cases resistant to first line SSA therapy. Few studies have reported using higher dose Pegvisomant or Combination of Pegvisomant and Pasireotide in these refractory cases with good effect (281,282). Use of radiotherapy has been utilized for control of the tumor volume in aggressive disease, while others have considered using the alkylating agent temozolamide or even cytotoxic therapy (222,283).

Management of Acromegaly in Pregnancy

Pregnancy in a healthy non-pregnant female is associated with gradual decline in pituitary derived GH levels, as the placental GH levels rise throughout the pregnancy. IGF-1 levels initially tend to decrease due to enhanced estrogen effect on liver, but eventually the levels rise as an action of placental GH. These physiological changes seem to explain the relatively less aggressive or rather benign course of acromegaly in pregnancy (284).

Infertility is more common in active disease, and patients often patients require treatment to achieve pregnancy. Evaluation of baseline tumor volume in a planned pregnancy is useful to safely plan monitoring during pregnancy. GH and IGF-1 levels are unreliable due to assay interference and are not routinely used to guide decision making (212). As a consequence of physiological changes, prevalence of gestational diabetes and hypertension is higher in women with acromegaly, but this seems to correlate with the pre-pregnancy control and not the degree of rise of IGF-1 (285). For patients with macroadenoma, serial visual field testing is required during pregnancy. Patients with intractable headaches, cranial nerve deficits or visual manifestations are likely to require intervention. If MRI is required it is best undertaken as unenhanced study (212). As GH does not cross the placenta, no direct effect on the disease on fetus have been reported. Studies have shown that the tumor size does not usually increase during pregnancy (286). The current recommendations suggest all medical therapy should be ceased at diagnosis of pregnancy. If pregnancy is pre-planned it is recommended that long acting SSA are discontinued about two months prior to pregnancy and patient switched to short acting octreotide injections (151,287). Safety data for the use of SSA and Pegvisomant is not substantial, but the available evidence has not shown any significant impact on maternal or fetal outcomes. There are reports of SSA use being associated with small for gestation babies, without malformations and similar concerns with use of dopamine agonists (285,288). While there are concerns for premature delivery the data for use of Pegvisomant in pregnancy is more encouraging (289).

NOVEL AGENTS IN THE TREATMENT OF ACROMEGALY

A number of novel agents are in advanced stages of development for the medical treatment of acromegaly. These include agents that continue to work by the somatostatin mechanism as well as new mechanisms of action. Developments in the understanding of the molecular pathogenesis of growth hormone excess and pituitary tumor development have led to the identification of novel targets for drug development. New treatments need to be safe and well tolerated, as well as effective and importantly cost effective.

An anti-sense oligonucleotide has been developed directed against the growth hormone receptor. Early clinical trial data suggests that this strategy may prove effective in reducing growth hormone signaling and IGF-1 generation in patients with acromegaly (290). The drug was well tolerated in an early clinical trial with injection site reactions the most common adverse event reported.

Novel compounds with combined affinity for SSTR2, SSTR5 and the dopamine D2 receptor are also being developed and in vitro show enhanced inhibition of growth hormone release (291). The ongoing development of these chimeric analogs may increase the efficiency of currently available analogs (292).

Somatoprim or Veldoreotide is a novel somatostatin analogue. This agent has affinity for the SST2, 4 and 5 receptors. A phase II study to investigate the efficacy of this agent in acromegaly is underway. STAT3 signaling is an important mechanism in the regulation of growth on dependent gene expression. GH-secreting adenomas overexpress STAT3. Recently a STAT3 inhibitor has been shown to suppress growth action. Thus, there is early evidence that this novel strategy may have a role in the treatment of acromegaly in the future (293). In addition, a new formulation of subcutaneous octreotide depot has been trialed in phase II studies, demonstrating superior efficacy to intramuscular octreotide (294).

In summary, continuing advances in the understanding of the mechanisms responsible for pituitary tumor development and the regulation of GH secretion, are aiding the further development of existing therapeutic agents and enabling the creation of new promising treatment for patients with acromegaly.

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Endocrine Changes In Obesity

ABSTRACT

Obesity can be associated with several endocrine alterations arising from changes in the hypothalamic-pituitary hormones axes. These include hypothyroidism, Cushing’s disease, hypogonadism, and growth hormone deficiency. Besides its role in energy storage, adipose tissue has many other important functions that can be mediated through hormones or substances synthesized and released by adipocytes, including leptin and adiponectin. Further, obesity is also a common feature of polycystic ovarian syndrome with hyperinsulinemia being the primary etiological factor. Here, we provide an overview of several endocrine syndromes known to result in obesity and discuss the endocrine role of adipose tissue in conjunction to its association with hypothalamic-pituitary-endocrine axes.

INTRODUCTION

This chapter will discuss the endocrine role of adipose tissue and how alterations in each of the hypothalamic-pituitary-endocrine axes can occur in association with obesity. Of particular relevance is the possible bidirectionality of the relationships between endocrine changes and obesity: whether they are secondary to obesity or, in some cases, be a contributive factor to the development and/or perpetuation of obesity.

The endocrine axes of the human body are dynamic systems; they frequently show changes in response to stress, disease, or other pathological states. For example, during acute and chronic illnesses, and low calorie or starvation states, levels of thyroid, gonadal, and growth hormone are altered, returning to normal as the subject recovers. These hormonal changes are, therefore, thought to be secondary to the disease state and their recovery is reflective of homeostatic responses. Often these "adaptive" changes in hormonal dynamics may not necessarily be appropriate. Likewise, therapeutic measures aimed at restoring "normal" serum level of perturbed hormones offered in hopes of hastening recovery and improve patient outcomes have generally not been shown to be beneficial.

The weight gain that leads to obesity is the consequence of a positive energy balance, which can result from an increased energy intake, decreased energy expenditure, or both. This misalignment may be thought of as a failure of the body's homeostatic mechanisms to match energy intake with expenditure. Different obesity phenotypes may have variable health implications. For example, abdominal obesity is considered a more hazardous condition than gluteofemoral, or gynecoid, obesity. In those with abdominal obesity, accumulation of intraperitoneal fat (omental and visceral fat) carries greater health risk than the subcutaneous compartment. Therefore, when discussing complications of and metabolic abnormalities associated with obesity, different obesity phenotypes are recognized to carry different degrees of cardiometabolic risk.

Our understanding of the physiology of adipose tissue has greatly advanced in the last decade and extensive research has been dedicated to the study of the interactions between the adipose tissue and other bodily systems, in particular the central nervous system. New hormones have been discovered with potentially important roles in energy balance and food intake. The roles of many of these newly discovered hormones have not been fully elucidated in humans, but the future holds promise in not only improving our knowledge of the pathophysiology of obesity but also in developing novel therapeutic approaches to complement our currently, rather limited, pharmacological arsenal.

ADIPOSE TISSUE AS AN ENDOCRINE ORGAN

Adipose Tissue

Adipose tissue has many important functions other than energy storage that are mediated through hormones or substances synthesized and released by adipocytes. These substances, termed "adipocytokines," act on distant targets in an endocrine fashion or locally in paracrine and autocrine fashions. In the following paragraphs, we shall discuss a few of the important adipocytokines secreted from “white" fat. For further characterizations of other types of adipose tissue, including "brown" and “pink” fat, see the Endotext chapter (1).

Leptin

The hormone leptin (from the Greek word ''leptos'' meaning ''thin'') is a 167-amino acid peptide hormone encoded by the ob (obesity) gene and secreted by white adipocytes. Its discovery in 1994, has greatly improved our understanding of how adipose tissue "communicates" with other systems in the body, in particular with the central nervous system (CNS). Following release into the circulation, leptin crosses the blood–brain barrier and binds to presynaptic GABAergic neurons of the hypothalamus of the CNS controlling appetite and energy expenditure (2). One of leptin’s key roles is thought to be as a signal of inadequate food intake or starvation. For example, leptin levels decline during fasting, low-calorie dieting, and uncontrolled type 1 diabetes. In these situations, the reduced leptin levels stimulate hunger while decreasing energy expenditure and engendering other physiologic adaptations that restore fat stores, and in turn leptin levels, to baseline (3,4).

On the other hand, serum concentrations of leptin increase in proportion to increasing adiposity. As a regulatory signal in a homeostatic system, if the leptin receptor is functioning normally, then higher circulating leptin levels should result in decreased energy intake and elevated energy expenditure, but this is not the case when individuals become overweight or obese. Instead, in patients with obesity high leptin levels are associated with low circulating soluble leptin receptors (SLR) consistent with a state of leptin resistance (5). Leptin must cross the blood–brain barrier (BBB) to reach the hypothalamus and exert its anorexigenic functions. Decreased transport across the blood-brain barrier (6)and a decreased ability of leptin to activate hypothalamic signaling in diet-induced obesity (7-9) may be crucial in the pathogenesis of leptin resistance.

In addition, anatomical and physiological changes that obesity can cause to the hypothalamus include expression of leptin signaling inhibitors, hypothalamic inflammatory signaling and gliosis, and endoplasmic reticulum stress.Elevated leptin itself may attenuate downstream leptin action, creating a functional ceiling for leptin action (10). These changes, together with the blood-brain barrier alterations, contribute to a failure of rising leptin levels to adequately compensate for the positive energy balance and thus promote the state of unwanted weight gain and obesity. Taken together, evidence points to leptin’s primary function as a defense against decreased body weight rather than to limit increases in body weight (10)

Data also suggests that leptin resistance can be a pre-conditioning factor contributing to diet induced obesity. Animal studies show that rats with a pre-existing reduction in leptin sensitivity develop excessive diet-induced obesity without eating more calories or altering their leptin sensitivity (11). This postulated leptin resistance is a major target in the search for a better understanding of obesity and the development of pharmacological tools to treat this chronic disease.

Most people with obesity are hyperleptinemic and show little or no weight loss after leptin treatment. However, recent evidence has indicated that a subset of patients with obesity have low endogenous plasma leptin levels and robustly respond to leptin treatment (12). These findings have led to a proposed classification of obesity based in leptin secretion and action. Type 1 obesity is associated with low leptin levels and leptin replacement can be an effective treatment in these forms of diabetes and obesity. Examples of patient populations in which this is more likely to be true include children with early onset and severe obesity (congenital leptin deficiency) (13) and those with generalized non-HIV lipodystrophy in whom recombinant methionyl human leptin has been FDA approved (14,15). Type 2 obesity is associated with leptin resistance, in which case leptin replacement is not optimal and other therapeutic approaches should be pursued (12).

Leptin plays a significant permissive role in the physiological regulation of several neuroendocrine axes, including the hypothalamic-pituitary-gonadal, thyroid, growth hormone, and adrenal axes (16,17). Leptin regulates reproductive function by altering the sensitivity of the pituitary gland to GnRH and acting at the ovary to alter follicular and luteal steroidogenesis, proliferation, and apoptosis (17). Thus, leptin serves as a putative signal that links metabolic status with the reproductive axis.

Leptin receptors are also present in peripheral organs, such as the liver, skeletal muscles, pancreatic beta cells, and even adipose cells, indicating endocrine, autocrine, and paracrine roles of leptin in energy regulation. Leptin signaling in these organs is thought to mediate important metabolic effects. For example, leptin has been implicated in glucose and lipid metabolism as an insulin-sensitizer (18). It has been shown to decrease glucagon synthesis and secretion, decrease hepatic glucose production, increase insulin hepatic extraction, decrease lipogenesis in the adipose tissue, and increase lipolysis among multiple other beneficial effects on insulin and lipids metabolism (19).

Other identified links between leptin and biological systems include expression of leptin by placenta and in fetal tissues. In this context, leptin is thought to be important for placentation, maternal-fetal nutrition, and stimulating hematogenesis and angiogenesis in the regulation of fetal growth and development (20). On the other hand, the pathological expansion of white adipose tissue during expression of obesity and subsequent increases in cytokines and leptin have been implicated in worsening local and systemic inflammation, sustained proliferative signaling, epithelial-to-mesenchymal transition, angiogenesis, and cellular energetics (21) in association with increased risk of endometrial, kidney, and breast cancers (21,22).

Adiponectin

Adiponectin is another important adipocytokine that influences insulin sensitivity and atherogenesis. Adiponectin mediates its effect through binding to receptors AdipoR1 and AdipoR2, leading to activation of adenosine monophosphate dependent kinase, PPAR-α, and other yet-unidentified signaling pathways (23). Lower levels of adiponectin in obesity have been associated with insulin resistance (24), dyslipidemia (25), and atherosclerosis (26) in humans. With weight loss, plasma adiponectin levels significantly increase in parallel with improvements in insulin sensitivity (27). In a study with 2258 children with overweight or obesity, independent of the degree of obesity, leptin, adiponectin, and the leptin/adiponectin (L/A) ratio were associated with insulin resistance and other cardiometabolic comorbidities (hyperglycemia and dyslipidemia), but the L/A ratio exhibited stronger associations than the respective adipokines (28).

Genetic analysis of single nucleotide polymorphisms (SNP) in the adiponectin locus have identified in humans a haplotype that, in presence of reduced adiponectin and obesity might alter metabolic profile posing risk towards type 2 diabetes. Presence of +10211T/G and +276G/T SNP are associated with increased fasting plasma glucose, body mass index (BMI), and hypertriglyceridemia (29). Recently, adiponectin was found to enhance exosome biogenesis and secretion, leading to a decrease in cellular ceramides, the excess of which is known to cause insulin resistance and cardiovascular disease phenotypes (30). Adiponectin has been shown to reduce the action of inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) (31), favorably modulate natural killer cell function (32) and other immune regulatory molecules (33), and improve dyslipidemia (34) and other risk factors of cardiovascular disease (31).

In addition to an anti-atherogenic effect, adiponectin may also have a variety of anti-tumor effects. This is thought to be mediated, in part, through inhibition of leptin-induced tumor proliferation (35). It retards the aggressiveness of tumors and their metastatic potential. By cancer site and type, high adiponectin levels are associated with a decreased risk of breast, colorectal, and endometrial cancer (22), whereas hypoadiponectinemia has been associated with increased risk for breast, gastric, lung, and prostate cancers (36-39).

A recent study also linked maintenance of the balance between adiponectin and leptin levels with cellular changes in human milk that enhances the protection and decreases the indices of neonatal infection in the breastfeeding infants of women with high BMI values (40).

Chemerin

Chemerin, also known as Retinoic Acid Receptor Responder Protein 2, is a newly discovered adipokine secreted from mature adipocytes thought to play an important role in the regulation of adipogenesis as well as macrophage infiltration into adipose tissue (41,42). Overexpression of chemerin in people with obesity correlates with early vascular damage, as chemerin was demonstrated to be a better predictor of intima-media thickening than waist circumference and glycated hemoglobin. Weight loss is associated with a decrease of chemerin level and, like adiponectin, an improvement of all parameters of the metabolic syndrome (43). Also, a decrease of chemerin is independently associated with the reduction of carotid intima-media thickening and the improvement of insulin sensitivity (44).

Omentin

Omentin is an adipokine preferentially produced by visceral adipose tissue that exerts insulin-sensitizing actions (45). Its expression is reduced in obesity, insulin resistance, and type 2 diabetes. Omentin is also positively related with adiponectin and high-density lipoprotein levels, and negatively associated with body mass index, waist circumference, insulin resistance, triglyceride, and leptin levels (46) (47). Apart from obesity, hyperandrogenism and PCOS per-se seem to have an additional role in omentin levels since omentin-1 was lower in girls with obesity, PCOS, and hyperandrogenism compared to girls with obesity but not PCOS (48).

Omentin has anti-inflammatory, anti-atherogenic, anti-cardiovascular disease, and anti-diabetic properties (46). Regarding its effects in the cardiovascular system, omentin causes vasodilatation of blood vessels and mitigates C-reactive protein-induced angiogenesis. The ability of omentin to reduce insulin resistance in conjunction with its anti-inflammatory and anti-atherogenic properties makes it a promising therapeutic/diagnostic target (49).

Omentin levels are not significantly different during pregnancy in mothers with diabetes compared to controls. However, significantly lower levels were observed in offspring of the mothers with diabetes, suggesting an increased risk for the development of insulin resistance in later life (50).

Retinol Binding Protein-4

Retinol binding protein-4 (RBP-4) belongs to the lipocalin family that transports small hydrophobic molecules and is produced primarily in the liver and mature adipocytes (51). Although the relationship between serum RBP-4 and obesity in humans has not been confirmed yet in population studies, several studies have shown positive correlations between the expression of RBP-4 and BMI and glucose concentration (52). RBP-4 levels can be reduced by weight loss, consuming a balanced diet, and exercise in association with increased insulin sensitivity (53,54).

Visceral Adipose Tissue-Derived Serpin: Serpin A12 (Vaspin)

Vaspin is a serine protease inhibitor produced by subcutaneous and visceral adipose tissue. Vaspin is also expressed in the skin, hypothalamus, pancreatic islets, and stomach. Vaspin is considered as an anti-atherogenic insulin-sensitizing factor (55).

Fatty Acid-Binding Proteins

Fatty acid binding protein A (A-FABP) is an isoform expressed in the adipose tissue and macrophages (56). It binds to hydrophobic ligands such as long chain fatty acids and facilitates their transport to specific cell compartments. Several studies have shown positive correlations between A-FABP and proinflammatory factors, such as CRP, and may also have significant importance in predicting insulin resistance (57).

Acylation Stimulating Protein

Acylation stimulating protein is synthesized and secreted by adipocytes and plays a major role in fatty acid uptake and triglyceride synthesis in these same cells, including postprandial clearance of triglycerides (58). It has been shown to induce glucose-stimulated insulin release from pancreatic beta cells, modulates cytokine synthesis by mononuclear cells, as well as inhibit cytotoxicity of natural killer cells (59).

Renin-Angiotensin-Aldosterone System

Several components of the renin-angiotensin system (renin, angiotensinogen, angiotensin-converting enzyme, and angiotensin 2 receptors) are expressed by adipose tissue (60). Recent studies have shown that adipocyte deficiency of angiotensinogen prevents obesity-induced hypertension in male mice (61). Adipocytes promote obesity-induced increases in systolic blood pressure in male high fat-fed C57BL/6 mice via angiotensin 2 dependent mechanism (62). Adipocyte angiotensinogen deficiency prevents high fat-induced elevations in plasma angiotensin 2 concentrations and therefore in systolic blood pressure (61). These results suggest that adipose tissue serves as a major source of angiotensin 2 in the development of obesity-related hypertension.

Other Factors Secreted by Adipose Tissue

Other proteins secreted by adipose tissue include plasminogen activator inhibitor-1 (PAI-1) (63) as well as complement factors adipsin, apelin, and pten, which may have roles in the pathophysiology or the progression of coronary artery disease and type 2 diabetes (64,65).

Circulating levels of Interleukin-6 (IL-6) are significantly higher in patients with overweight and obesity (66). Interleukin-6 is released by macrophages and T-cells in the adipose tissue (67) and has been implicated in regulating insulin signaling in peripheral tissues by promoting insulin-dependent hepatic glycogen synthesis and glucose uptake in adipocytes (68). Recent studies show that IL-6 deficient mice develop late-onset obesity as well as disturbed glucose metabolism (69). The mechanisms underlying the effect of IL-6 on body fat and metabolism are not completely understood. However, IL-6 may exert central effects to decrease fat mass because of increased energy expenditure. Administration of IL-6 to the CNS has, for instance, been shown to induce energy expenditure and reduce fat mass more effectively than peripheral treatment (69). It has been suggested that IL-6 potentiates the action of leptin providing a possible mechanism for its anti-obesity effect (70). In addition, IL-6 has been postulated to play an etiologic role in the increased risk of thromboembolism observed in patients with obesity (71).

Summary

Adipose tissue is an extremely active organ with multiple roles, including endocrine, paracrine, and autocrine, in human physiology and disease. How these roles are performed and their contribution to the health or risk of disease will likely be elucidated as more discoveries continue to shed light on the mechanism of the complex interaction between adipocytes and other body tissues.

OBESITY AND HYPOTHALAMIC-PITUITARY AXES

Obesity and Sex Hormones

Not only is obesity associated with alterations in sex hormone levels but sex hormones may conversely influence expression of different obesity phenotypes. One of the best examples of this is the relationship between obesity and androgen levels in men and women and the roles played by sex hormone-binding globulin (SHBG) and gonadotropins (72-74).

SEX STEROID AND SHBG

Most circulating testosterone and estrogen are bound to proteins, SHBG and albumin. Although a portion of the bound sex hormones may be available for use by the body target cells, only about 2% of circulating sex steroids are unbound, or free, and constitute the bioactive fraction of these hormones. Total hormone levels, therefore, reflect the bound and unbound hormone and are greatly dependent on the serum concentration of SHBG. For example, SHGB levels increase with age and bioactive testosterone levels decrease (Table 1).

Table 1. Common Conditions and Medications that Affect Serum Concentrations of SHBG

Increased SHBG

Decreased SHBG

Older Age

Cirrhosis

Hyperthyroidism
Estrogens

Obesity Androgens
Hypothyroidism
Glucocorticoids
Growth hormone
Insulin

OBESITY AND ANDROGENS IN MEN

Testosterone should be measured in the morning when serum concentrations peak and we recommend repeating an abnormal measurement for confirmation. Evidence indicates that testosterone (T) deficiency in men induces adiposity and, at the same time, increased adiposity induces hypogonadism (72). An obesity-associated decline in SHBG might partially explain the observed fall in T levels (74,75). However, an increased BMI is associated with low measured, or calculated, free- and bioavailable-testosterone levels as well. In a metanalysis of sixty-eight studies including a total of 19,996 patients with obesity, prevalence of hypogonadism ranged from 22.9 to 78.8% and from 0 to 51.5% depending on whether low total testosterone or low free testosterone was used to define hypogonadism, respectively. Pooled prevalence of hypogonadism when measuring total testosterone or free testosterone was 42.8% and 32.7%, respectively (76).

While the specific pathogenic mechanisms linking obesity with low testosterone levels are not completely understood, both secondary (hypogonadotropic) and, to a minor degree, primary hypogonadism (testicular failure) have been described. Other potentially contributing factors include development of type 2 diabetes, hypertension, and increased adipokines (77,78). Obstructive sleep apnea predisposes to male obesity and secondary hypogonadism (MOSH) through reductions in luteinizing hormone (LH) pulse amplitude and reduced mean serum levels of LH and T in men. Obstructive sleep apnea may also disrupt the association between a rise in serum T levels and the appearance of first REM sleep (79,80).

At the testicular level, studies by Wagner et al have shown that obesity lowers the number of testosterone producing Leydig cells and promotes destruction of existing ones by increasing levels of proinflammatory cytokines (TNF alpha) and cells (macrophages) (81). In both the short and long term, obesity was shown to lower intra testicular levels of testosterone by way of increasing serum leptin and estradiol levels and inhibiting the expression of the gene for cytochrome p450 of the cholesterol side chain cleavage enzyme (Cyp11a1) (81).

Whether testosterone treatment in (MOSH) is beneficial has long been controversial. Only those with low free T levels and signs or symptoms of hypogonadism should be considered androgen deficient. Considering the limited number of rigorous testosterone therapy trials that have shown beneficial effects, the modest amplitude of these effects, and unresolved safety issues, testosterone therapy is currently not advocated in the prevention or reversal of obesity-associated metabolic disturbances (82).

On the other hand, true hypogonadism in men can promote increased fat mass, which in turn may worsen the hypogonadal state. Low testosterone levels lead to a reduction in muscle mass and an increase in adipose tissue within abdominal depots, especially visceral adipose tissue (VAT) that can be reversed with testosterone therapy (83,84). As adiposity increases, there is a further raise in aromatase activity that is associated with an even greater conversion of T to estradiol (often termed the 'testosterone-estradiol shunt'), which is thought to decreased GnRH secretion (85). This further decreases T levels that in turn further increases the preferential deposition of fat within abdominal depots: a 'hypogonadal-obesity cycle' (86,87). Individuals with obesity retain the capacity to reverse this gonadotrophic response with weight loss, demonstrating that MOSH is a reversible condition. This has been made evident on several studies in which weight loss normalized T levels (88,89).

In summary, obesity is frequently associated with low androgen levels in men and true hypogonadism can worsen adiposity and central fat deposition. The pathogenesis of obesity-related hypogonadism is complex and multifactorial, implicating obesity-related comorbidities and changes in body fat mass itself with its multiple adipokines and inflammatory mediators. Ultimately, these changes are frequently reversible with weight loss and preferred strategies to manage these conditions target lifestyle, anti-obesity medications, and weight-loss surgeries when indicated.

OBESITY AND SEX STEROIDS IN WOMEN

Increases in body weight and fat tissue are associated with abnormalities of sex steroid levels in both premenopausal and postmenopausal women. It has been shown that women with central obesity have higher circulating androgen levels, even in the absence of a clinical diagnosis of polycystic ovarian syndrome (PCOS) (90,91). These women have higher total and free testosterone levels than normal-weight woman and lower androstenedione and SHBG levels (91). Some studies examining the co-relationships between the total testosterone levels and phenotypic features of hyperandrogenism, such as hirsutism, found a strong correlation between them, regardless of the assay used for assessment (92).

The timing of menarche is primarily thought to be affected primarily by genetic factors (93,94), but the average age at menarche in US girls has been declining over the past 30 years (95) in conjunction with changes in nutritional status (96). A Mendelian randomization study from the United Kingdom linked a higher BMI with early menarche, suggesting a causal relationship between increasing prevalence of childhood obesity and similar trends in the prevalence of early menarche (97).

Studies have also shown that the earlier the onset of menarche, the higher the risk of developing obesity (98) and other comorbidities in the adult life, independently of BMI, such as: breast cancer, cardiovascular disease, cerebrovascular disease, type 2 diabetes, and adolescence at-risk behaviors (99-104). Consequently, all-cause mortality has been linked with early menarche (105). Also, there is evidence that menarche at or before 12 years of age is associated with higher androgens levels even during adulthood, suggesting that hyperandrogenemia may explain, at least in part, the higher incidence of comorbidities among these women. A recent study demonstrated that with each one-year advance in menarcheal age, the probability of having obesity decreased by 22%; interestingly, in this study women with obesity had higher androgens levels (106).

Menarche age also appears to affect offspring. Boys whose mothers with menarche onset ≤13 years at menarche had an adjusted relative risk of obesity 3-fold greater than sons of mothers with a later menarche onset. The increased obesity risk was not observed in daughters. However, girls who experienced menarche earlier had a less favorable anthropometric profile consisting in a reduced waist and hip circumferences and waist-to-height ratio (107). Early menarche, therefore, has emerged as a risk of later obesity and related medical problems.

RELATIONSHIP BETWEEN LEPTIN AND SEX HORMONES

Leptin participates in the regulation of hypothalamus-pituitary-gonadal (HPG) axis at multiple levels. Leptin appears to facilitate GnRH secretion indirectly by modulating several interneuron secretory neuropeptides (108,109) and directly by stimulating LH and, to a lesser extent, FSH release.

Leptin has a permissive role in timing puberty but is not essential nor is the only trigger for puberty onset, as has been shown in studies (110) of patients with leptin deficiency and several animal studies (111,112).

Kisspeptin play a central role in the modulation of GnRH pulse generator and, thus, downstream regulation of gonadotropins and testosterone secretion in men (113,114). Kisspeptins are mostly distributed in the hypothalamus, dentate gyrus and adrenal cortex. Inactivating mutations of the kisspeptin receptor have been shown to cause hypogonadotropic hypogonadism in men, while an activating mutation is associated with precocious puberty. Data from studies in animals link kisspeptin expression with hyperglycemia, inflammation, leptin and estrogen, factors known to regulate GnRH secretion. It has been hypothesized that decreased endogenous kisspeptin secretion is the common central pathway that links metabolic and endocrine factors in the pathology of T deficiency observed in MOSH and type 2 diabetes (113).

Serum kisspeptin levels are higher in patients with obesity and tend to decrease after weight loss intervention. (115,116). Also, data suggest a higher concentration of serum kisspeptin in women with PCOS irrespective of their BMI but further data are needed to ascertain the role of kisspeptin in PCOS (116).

Kiss1 neurons appear to transmit the regulatory actions of metabolic cues on pubertal maturation. Recently, it has been documented that AMPK and SIRT1 operate as major molecular effectors for the metabolic control of Kiss1 neurons and, thereby, puberty onset. Alterations of these molecular pathways may contribute to the perturbation of pubertal timing linked to conditions of metabolic stress in humans, such as undernutrition and obesity. As such, it has the potential of becoming a druggable targets for better management of pubertal disorders (117).

Leptin receptors are also widely expressed in the human ovaries (118) and testes (119) indicating a direct gonadal regulatory role. Studies by Ma et al. have shown that high-fat diet fed mice produce fewer oocytes compared with control mice receiving a normal diet. Leptin has been noted to act locally within the mice ovarian granulosa cells to reduce estradiol production (120). These actions are mediated via induction of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the granulosa cells (GCs), which in turn detrimentally affects intermediate steps of estradiol synthesis including, intracellular cAMP levels, MAPK signaling, and aromatase mRNA expression (121). In humans undergoing in vitro fertilization, Ma et al. demonstrated that subjects with higher BMI had higher levels of CART mRNA and peptide in follicular fluid (121). Therefore, in women with obesity, evidence supports a role for leptin as a mediator of infertility at the level of the ovary.

As mentioned above, in men with obesity, intra testicular levels of testosterone are lower due to leptin and estradiol inhibition of the expression of the gene for cytochrome p450 of the cholesterol side chain cleavage enzyme (Cyp11a1) (81). Gregoraszczuk et al exposed porcine ovarian follicles obtained from prepubertal and mature animals to progressively increasing doses of super active human leptin antagonist (SHLA) and measured levels of leptin receptor (ObR), leptin, CYP11A1 and 17β-hydroxysteroid dehydrogenase (17β-HSD), progesterone (P4), and testosterone (T) in the follicles (122). These experiments showed that SHLA inhibits CYP11A and 17 beta protein expression, subsequently inhibiting leptin, ObR, and hence leptin-mediated follicular P4 and T secretion. Women with obesity and polycystic ovarian syndrome (PCOS), a condition associated with elevated androgen levels and infertility (see also below), were found to have higher levels of leptin (both bound and free form) and lower levels of s-OBR (soluble Leptin receptors) when compared to lean females with PCOS, after adjusting both groups for age, in studies by Rizk, who hypothesized that lower s-OBR may have been in response to impaired leptin function (123).

Leptin and its soluble receptor are thus implicated in the pathophysiology of PCOS, may act as a mediator of infertility at the level of the ovary and testes, and that leptin antagonists acting peripherally in gonadal tissues may thus be useful in modifying the physiology of reproduction.

OBESITY AND POLYCYSTIC OVARIAN SYNDROME

Polycystic ovarian syndrome is a highly prevalent condition of hyperandrogenism frequently associated with obesity. Hence, this disorder has been studied extensively in the context of interactions between sex hormones and obesity. It affects approximately 6-10% of women in reproductive age (124). About two thirds of women with PCOS are obese and 50-70% of them have insulin resistance (IR) (125).

Adult men have more visceral fat than premenopausal women, in which the body fat is more prominent in the periphery and subcutaneous adipose tissue. This sexual dimorphism is mainly related to the differential effects of androgens and estrogens on adipose tissue (126). Visceral adipose tissue (VAT) excess is strongly associated with metabolic disorders such as insulin resistance and dyslipidemia (127). Women with PCOS manifest what has been called "masculinization of the adipose tissue" characterized by increased VAT and even male pattern adipokine gene expression with its associated metabolic complications (128,129). Even though increased VAT plays a significant role in the development of insulin resistance in PCOS, it has been suggested that insulin resistance may represent an intrinsic characteristic of this syndrome, independent of obesity (124). Interestingly, in PCOS, despite the insulin resistance in other organs, the ovaries remain sensitive to the stimulatory effect of insulin on androgen production (130). A recent study showed that despite women with PCOS and women with the metabolic syndrome sharing many features, these are different entities, mainly due to the excess of androgens seen in PCOS, which seems the be the main culprit of its multiple co-morbidities (131) .

Anovulation and menstrual irregularities are major features of PCOS in part due to ovarian hyperandrogenism, hyperinsulinemia due to IR, and altered paracrine signaling within the ovary, which can disrupt follicle growth (124). Hyperinsulinemia also decreases hepatic SHBG with a subsequent increase in free androgens levels. In addition, insulin increases the androgens synthesis stimulated by LH and IGF-1.

An increased ratio of serum LH to FSH may be seen in about 70% of women with PCOS (132,133). The androgen excess reduces the negative feedback in the hypothalamus causing an enhanced pulsatile release of gonadotropin releasing-hormone (GnRH) which will elevate LH levels and pulse frequency (134).

In summary, obesity is a common feature of PCOS and hyperinsulinemia secondary to insulin resistance of the liver and muscle is believed to be the main etiological factor behind the development of PCOS. Obesity also leads to hyperestrogenism. Weight loss and/or use of insulin sensitizing agents (mainly metformin) improve insulin sensitivity, reduce insulin levels, and improve fertility in women with PCOS but not live births (135,136). Therefore, the role of metformin in improving reproductive outcomes in women with PCOS appears to be limited (137). Letrozole, an aromatase inhibitor, is headed toward replacing clomiphene, a selective estrogen receptor modulator. As the first-choice option for ovulation induction, metabolic treatments such as metformin, troglitazone, or d-chiro-inositol have failed to show promise in improving fertility outcomes. Further studies are needed of the newer agents to treat type 2 diabetes (138) .

A clinical trial in 120 infertile PCOS women showed that when metformin is combined to myoinositol (MI) a significant improvement in live birth rate, menstrual cycle (length and bleeding days), and HOMA index is observed compared to use of metformin alone (139). Treatment with MI has been useful also in in-vitro fertilization (IVF), as it allows a decrease in the amount of recombinant FSH administered, in the duration of the ovulation induction for follicular development (140,141) and an increase in the clinical pregnancy rate (142) [45].

OBESITY AND ESTROGENS

Estrogens play an important role in body weight, fat distribution, energy expenditure, and metabolism. In healthy premenopausal women, estrogens are mainly synthesized in the ovaries under the regulation of gonadotropins releasing hormones from the pituitary gland. They are also produced in the adipocytes via aromatization from androgenic precursors, which is especially important in men and post-menopausal women and increase in proportion to the total body adiposity (143,144).

Most metabolic effects of estrogens are mediated through estrogen receptor (ER) alpha, whereas most gynecologic actions are exerted through ER beta. Mice of both sexes with a targeted deletion of the ER alpha gene manifest obesity-induced insulin resistance with altered plasma adipokines and cytokines levels and increased adiposity, mainly VAT (145,146).

Estrogens have a positive effect in glucose homeostasis, acting as an insulin sensitizer at multiple levels, including skeletal muscle, liver. and adipocytes (147). Estrogen effects the immune system to decrease inflammation, thus favoring insulin sensitivity (148,149). Pancreatic islet-cells also have estrogens receptors, which when activated improve beta cell function and survival (150). Estrogen deficiency promotes metabolic dysfunction predisposing to obesity, metabolic syndrome, and type 2 diabetes.

In rodent models, estrogen has been shown to influence energy intake and energy expenditure via hypothalamic signaling. Estrogen receptor alpha is widely expressed in the ventromedial hypothalamus (VMH), area of the brain that controls food intake, energy and body weight homeostasis. In animal models, the lack of ER alpha in the VMH causes dramatic changes in energy balance leading to increased adiposity (147).

The gynecoid body fat distribution, characterized by increased fat depots into the subcutaneous tissue favoring gluteal/femoral areas and decreased VAT is mediated mainly by estrogens (147). Visceral fat is augmented in hypoestrogenic states, as seen in menopause. These changes in body fat composition can be prevented by estrogens replacement (151). Also, estrogen treatment of male-to-female transsexuals significantly increases fat deposition in all subcutaneous fat depots, while having little effect on the visceral fat compartment (152).

Obesity in both men and women is associated with elevated estrogens levels that result from aromatization of androgens in adipocytes (86). Increased adiposity is a known risk factor for the development and progression of breast cancer and this hyperestrogenic state is associated with increased risk of cancer (153), while weight loss improves prognosis of patients diagnosed with breast cancer and the reduction in estrogens levels may be, at least in part, responsible for this finding (154).

Obesity and Growth Hormone

Growth hormone (GH) is secreted by the pituitary gland. Most of GH-promoting effects are mediated by Insulin- like Growth Factor-1 (IGF-1), but GH also has effects independent of IGF-1. Serum IGF-1 concentrations represent the most accurate reflection of growth hormone biologic activity. The liver is the major, but not exclusive, source of IGF-1. About 50% of circulating growth hormone is bound to binding proteins. These include a high affinity Growth Hormone Binding Protein (GHBP), which represents the extracellular portion of the GH receptor. IGFs are mostly bound to IGF- Binding Proteins (IGFBPs) with IGF-1 is bound to IGFBP3.

Together GH and IGF-1 influence lipids, protein, and glucose metabolism so as to inhibit fat accumulation, promote protein accretion, and alter energy expenditure and body fat/muscle composition. Normally, GH secretion is suppressed as insulin increases in the postprandial period, which permits skeletal muscle glucose uptake promoting glycogenesis and adipogenesis (155). The opposite changes in hormonal concentrations occur during fasting to facilitate lipolysis and hepatic glucose output (156).

GH secretion from the anterior pituitary is modulated by the hypothalamic GH releasing hormone (GHRH) and follows a pulsatile pattern that is influenced by age, sex, sleep, feeding, physical activity and weight (157). Obesity is typically accompanied by a decrease in GH levels and increase in GHBP levels. This is the opposite picture to starvation in which GH levels are increased and GHBP levels decreased. An inverse relation exists between GH levels and BMI and percent fat mass, particularly VAT, independently of age or sex (158,159). The reduction in GH levels in obesity is multifactorial and it involves a decreased pituitary release of GH (decreased frequency of GH secretory bursts proportionate to the decree of obesity) and an accelerated GH metabolic clearance rate (160).

Since GH has lipolytic and anabolic properties, it has been postulated that the decline of GH seen in elderly and individuals with obesity may be partly responsible for the progression of metabolic diseases (161). GH is known to induce insulin resistance (IR). The increased IR seen during puberty and gestational diabetes is, in part, attributed to increased GH action (162). One of the clinical manifestations of acromegaly is glucose intolerance and diabetes mellitus. But interestingly, GH deficiency can also be accompanied by increased IR. A recent general population study in Danish adults revealed that both low and high-normal IGF-1 levels are related to IR (163). There are striking similarities between the metabolic syndrome and untreated adult-onset GH deficiency: increased VAT, IR, non-alcoholic fatty liver disease, dyslipidemia and the associated increased risk of premature atherosclerosis and cardiovascular disease (164,165). All these observations have led to an increasing interest in investigating the mechanisms behind the decline of GH seen in obesity since it may have important clinical and therapeutic implications. Weight loss is associated with improved stimulated GH response. However there is uncertainty on how much weight loss is required to completely normalize GH secretion (166).

Despite the reduced GH levels seen in obesity, IGF-1 serum levels are not significantly different between those with and without obesity. Studies have reveled mostly normal or slightly low IGF-1 serum levels in individuals with obesity (159,167,168). This suggests that lower levels of GH are accompanied by increased peripheral sensitivity to GH accounting for the relatively normal IGF-1 levels. This is supported by data from Maccario et al., who found that the administration of a low dose of rhGH had an enhanced stimulatory effect on IGF-1 secretion in subjects with obesity compared to normal weight subjects (169). In another study, the same authors showed a normal feedback inhibitory response of the somatotroph to IGF-1 (170). In addition, decreased GH levels result in up-regulation of GH receptors and increased sensitivity at the liver, as it was shown by higher IGF-1 response to a single GH bolus in subjects with obesity as compared with normal weight individuals (171).

PROPOSED MECHANISMS FOR LOWER GH SECRETION IN OBESITY

Hyperinsulinemia that accompanies obesity could be one of the stronger inhibitors of GH secretion by peripheral and central actions. Insulin produces increased peripheral sensitivity to GH, reduced IGFBP-1 levels and increased IGF-1 in spite of decreased GH secretion by the somatotroph. High free IGF-1 levels in this case exert a negative feedback mechanism on GH secretion. Central effects of insulin were shown in a study where the peak GH secretion after GHRH stimulation was inversely associated with fasting insulin in premenopausal women with obesity (172).

Sex steroid levels may also govern GH activity. It has been shown that testosterone activates the somatotrophic axis in men (173,174) and augments the GH-dependent stimulatory effect on IGF-I production, enhancing protein and energy metabolism (175). Estrogens, in contrast, cause GH resistance in the liver, leading to a relative reduction of IGF-I production per unit of GH secretion (176).

Other possible mechanisms for the altered GH response in obesity are free fatty acids (FFA) and leptin, both of which are increased in obesity. Lee et al showed that reduction in free fatty acids concentrations in subjects with obesity through use of Acipimox leads to increased GH response to GH-releasing hormone (177). In animals, leptin has an inhibitory role on GH secretion from the pituitary gland through its effects on GHRH and neuropeptide Y (NPY) at the hypothalamus level (178).

RECOMBINANT GROWTH HORMONE THERAPY IN PATIENTS WITH OBESITY

The use of recombinant human growth hormone (rhGH) in elderly and subjects with visceral obesity results in several mild to moderate anthropometric and metabolic effects such as reduced fat mass, increased lean mass, and improved surrogate markers of cardiovascular disease (179). Recombinant growth hormone has been extensively studied as a treatment for obesity. A meta-analysis found that rhGH therapy reduces visceral adiposity and increases lean body mass as well as having beneficial changes in lipid profile in adults with obesity, but without inducing significant weight loss. In fact, the observed reductions in abdominal fat mass are modest and similar to what can be achieved by life style interventions (180). In addition, administration of rhGH was associated with increases in fasting plasma glucose and insulinemia over shorted periods of time (181). However, the dose of rhGH used in these studies was supraphysiological.

Investigations of rhGH in youth have reported favorable outcomes. A pilot study in young adults (18-29 years old) with obesity and non-alcoholic fatty liver disease suggested that rhGH may have benefits to reduce liver fat content (182). Also, in boys with obesity (8-18 years old) treatment with rhGH for one-year reduced body mass index standard deviation scores and insulin-like growth factor 1 levels increased. GH treatment also reduced low density lipoprotein cholesterol, total cholesterol, triglycerides, and alanine aminotransferase when compared with the baseline. (183). However, further studies of longer duration outcomes, including cardiovascular morbidity and insulin sensitivity, are warranted.

In conclusion, obesity is accompanied by a reduction in basal and stimulated GH secretion by the pituitary gland. The reduction in GH does not appear to translate into similar reduction in IGF-1. While some benefits of GH treatment in obesity are seen in body composition, other than in those individuals with documented GH deficiency, these are probably not enough (or greater than was is seen with lifestyle) to outweigh potential long-term side effects and the role of GH replacement in patients with obesity and normal GH axis testing, remains controversial.

Obesity and Adrenal Glands

Cortisol circulates in the bloodstream mainly bound to Cortisol-Binding Globulin (CGB or transcortin) and less to albumin. About 10% of cortisol is free or unbound and this fraction represents the bioactive portion of the hormone. CBG concentrations can be increased or decreased in several conditions and by some medications (Table 2), thus affecting total cortisol levels in these situations.

Table 2. Medical Conditions and Drugs that Affect Cortisol Binding Globulin (CBG) and Total Cortisol Levels
Increase CBG	Decrease CBG
Estrogens Pregnancy Oral contraceptives Diabetes mellitus Hyperthyroidism	Obesity Cirrhosis Testosterone Nephrotic syndrome Hypothyroidism

The dynamics of the hypothalamic-pituitary-adrenal (HPA) axis in obesity have been examined. Patients with Cushing's syndrome display several clinical features that resemble those seen in patients with the metabolic syndrome. These features include redistribution of adipose tissue from peripheral to the truncal region increasing VAT, insulin resistance, impaired glucose homeostasis, hypertension, and lipid abnormalities. These similarities led to the hypothesis that a dysregulation of the HPA axis in the form of "functional hypercortisolism" could potentially be a cause for abdominal obesity and its accompanying metabolic consequences (184).

The serum concentrations of cortisol are generally normal in obesity (185-188). Salivary cortisol and 24-hour urine free cortisol (UFC) excretion are usually high-normal or sometimes mildly elevated in obesity. A cross-sectional study of subjects with obesity showed a trend to increase salivary cortisol as BMI increased, but the same association was not found with UFC (189). Other studies in which UFC has been shown to be increased in obesity are due to enhanced cortisol clearance (188,190), with maintenance of normal cortisol levels and circadian appearance in those with obesity through subsequent increases in cortisol production rates (188,190,191).

It has been demonstrated that high-normal ACTH and cortisol levels in individuals with obesity are associated with cardiovascular risk factors, such as hypertension, insulin resistance and dyslipidemia (192,193). On the other hand, depression and/or alcoholism may slightly increase cortisol levels. These conditions have been described as pseudo-Cushing's syndrome (194). A pseudo-Cushing's state is characterized by clinical and biochemical features that resemble true Cushing's syndrome but with resolution of the signs and symptoms once the underlying primary condition is eliminated. It is thought that these primary conditions may stimulate CRH release with subsequent activation of the entire HPA axis (195,196).

Although serum cortisol is not increased in obesity, it is possible that the local production of cortisol in the fat tissue is increased and this, in turn, could lead to increased local action of cortisol with the subsequent metabolic consequences. Adipose tissue is involved in the metabolism of cortisol through action of the enzyme 11 Beta-hydroxysteroid dehydrogenase-1 (11HSD1), which converts cortisone (inactive corticoid) to cortisol (active corticoid) (197). Whole body 11β-HSD1 reductase activity tends to be higher in obesity (~10%) and is further increased by insulin (198). It appears that in obesity, more cortisol is derived from cortisone due to the increased activity of this hormone, which could simply be due to increased visceral fat mass. (198).

Some authors provide evidence that cortisol affects zinc metabolism and indicate possible repercussions on insulin signaling that might contribute to the development of resistance to the actions of insulin in obesity. Thus, alterations in the biochemical parameters of zinc observed in individuals who are obese contribute to the development of disorders in the synthesis, secretion, and action of insulin (199).

Visceral adipose tissue has higher numbers of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) than subcutaneous tissue (200,201). Glucocorticoids have higher affinity to MR than to GR. It has been shown that MR activation mediates inflammation and dysregulation of adipokines causing insulin resistance and acceleration of the development of metabolic disorder (202). Interestingly, blockade of the MR improves these outcomes (203,204). In human adipose tissue, MR mRNA levels increase in direct association with BMI and this augmentation is more significant in VAT, whereas GR mRNA levels had no apparent correlation with BMI or fat distribution (201). Even though evidence for an increased cortisol concentration within the VAT in human obesity is "possible, but unlikely" (205), it is not surprising that inhibition of 11HSD1 and MR has become a major therapeutic target in metabolic syndrome (206,207).

The cortisol response to a variety of stimuli such as ACTH, CRH, or meal ingestion is altered in obesity and by sex. Animal studies showed that estrogens sensitize and androgens diminish corticotropic-response to ACTH (208). In obesity these sex hormone differences are blunted. One study showed decreased ACTH potency with higher BMI in men (208) and other studies demonstrated ACTH secretion rates comparatively higher than the cortisol secretion rate in centrally obese premenopausal women; suggesting decreased responsiveness of the adrenal gland to the ACTH stimulation in these subjects (209,210). The same authors showed in a more recent publication, that premenopausal women exhibit diminished ACTH efficacy (maximal cortisol response) and sensitivity (slope of the dose-response curve) (211). This pattern is similar to what has been described in Cushing's syndrome (212). Of note, it is important to mention that older studies have revealed increased responsiveness of adrenal glands to exogenous ACTH pharmacologic stimulation (213), but this finding should not be extrapolated to the effects of endogenous ACTH stimulation.

A decrease in the mineralocorticoid receptor (MR) response to circulating corticosteroids was suggested by Jessop et al as an explanation for the relative insensitivity to glucocorticoid feedback in obesity (214). A more recent study showed that MR represent an important pro-adipogenic transcription factor that may mediate both aldosterone and glucocorticoid effects on adipose tissue development. Mineralocorticoid receptor thus may be of pathophysiological relevance to the development of obesity and the metabolic syndrome (215).

The HPA axis is also activated in response to stress along with the sympathetic nervous system, and the sympathoadrenal system. Whether stress-related obesity due to excess and/or dysfunction of cortisol activity is a distinct medical entity remains unclear and there are contradicting findings in the literature. This topic is evidently difficult to investigate due to multiple confounding variables and therefore well-defined longitudinal studies are needed (216).

Finally, when screening overweight and individuals with obesity for Cushing's syndrome it is imperative to follow the Endocrine Society guidelines which recommend diagnosing the disorder only if two screening tests are abnormal (196). A study of 369 overweight or subjects with obesity with at least two features of Cushing's syndrome found that 25% of these subjects had an abnormal screening test result, but none of them had two positive tests, hence none was found to have Cushing's syndrome (217).

In conclusion, obesity is associated with alterations in the HPA axis that may be a manifestation of a causative effect, adaptive changes to a new homeostatic state or, most likely, a combination of both. And although signs and symptoms of hypercortisolism commonly are also found in patients with central obesity, the finding of an actual case of Cushings disease is very rare in the obese population.

Obesity and the Thyroid

More than 99% of T4 and T3 circulate bound to transport proteins. Only a very small amount, less than 1%, of thyroid hormone is unbound or free and represents the biologically active fraction of the hormone. Thyroxine Binding Globulin (TBG) is the major transport protein for thyroid hormones and serum TBG concentrations are influenced by several conditions and medications, which result in altered total T4 and T3 concentrations (Table 3). Therefore, when evaluating thyroid function, we measure thyroid stimulating hormone (TSH) and free T4 (FT4). Free T3 (FT3) can also be measured in selected circumstances, such as hyperthyroidism, although it represents only a small fraction of circulation total thyroid hormone activity.

Table 3. Medical Conditions and Drugs that Affect Thyroxine Binding Globulin (TBG) and Total Thyroid Hormone Levels
Increase TBG	Decrease TBG
Estrogens Pregnancy Hypothyroidism Acute hepatitis	Androgens Corticosteroids Systemic illness Nephrotic syndrome Hyperthyroidism Cirrhosis

Thyroid dysfunction is frequently associated with changes in body weight and composition, body temperature, energy expenditure, food intake, glucose, and lipids metabolism. Hypothyroidism is linked to weight gain and decreased metabolic rate but there is also a positive association across the normal range between serum levels of TSH and BMI. Some cross-sectional population studies suggest that even a slightly elevated serum TSH might be important in determining an excess of body weight and it can be considered a risk factor for overweight and obesity (218-221). Also, individuals with obesity have an increased incidence of subclinical and overt hypothyroidism. Some studies showed a prevalence of these conditions in morbid obesity as high as almost 20% (222,223). Thyroid-stimulating hormone concentrations has also been associated with the presence of the metabolic syndrome, even when TSH is within normal levels. In a study of 2,760 euthyroid young woman, those with high-normal TSH (2.6-4.5 mIU/L) had higher prevalence of metabolic syndrome than those with low-normal TSH (0.3-2.5 mIU/L) (224) .

However, further investigation is needed to determine whether the relationship between TSH and BMI represents causality (mild thyroid failure leading to obesity) or just adaptive changes (physiologic or pathologic) to a new homeostatic state of increased body weight. Contradicting results from different studies illustrate this controversy. For example, a study published by Marzullo et al. supports the idea that obesity increases susceptibility for thyroid autoimmunity, since in their group of individuals with obesity they found higher rates of positive anti-thyroid peroxidase antibodies than in controls (225). This finding was not observed in other cross-sectional studies that included individuals with severe obesity (BMI > 40 kg/m²). In that study, as compared with controls subjects with severe obesity had higher levels of TSH (but with lower rates of positive thyroid antibodies than control individuals (222,223,226). Data from the NHANES III survey showed no difference in thyroid antibodies positivity among individuals with obesityand the general population (227). However, a recent metanalysis showed that even after stratification, the obese population had increased risks of overt hypothyroidism and subclinical hypothyroidism and was clearly associated with Hashimoto’s thyroiditis but not Graves' disease. In patients with Hashimoto’s thyroiditis, obesity was correlated with positive thyroid peroxidase antibody (TPOAb) levels but not with positive thyroglobulin antibody (TGAb) levels (228).

In a euthyroid population, when comparing metabolically healthy obese (MHO) with metabolically unhealthy obese (MUO) phenotypes, the following findings were reported: FT4 levels were negatively associated with the MUO phenotype, FT3 levels were positively associated with both the MHO and the MUO phenotypes, and TSH levels were positively associated with the metabolically unhealthy, non-obese phenotype (229).

Also, in population studies higher levels of T3, FT3, T4, and TSH are seen in individuals with obesity, probably the result of the reset of their central thyrostat at higher level (223).

The idea that these thyroid function tests (TFTs) changes may reflect a state of thyroid hormone resistance has also been considered. This is supported by the observation of decreased thyroid hormone receptors in circulating mononuclear cells of individuals with obesity (230) and decreased negative feedback between TSH and peripheral T3 levels.

Fat accumulation increases in parallel with TSH and FT3 levels independently of insulin sensitivity and other metabolic parameters. Also, a positive association has been described between FT3 to FT4 ratio and BMI and waist circumference (231). These findings may result from a high conversion of T4 to T3 due to increased deiodinase activity in the adipose tissue as a compensatory mechanism to increase energy expenditure (220). On the other hand, during a hypocaloric diet, serum T3 declines significantly, generating changes in the cardiovascular system like those seen in hypothyroidism, suggesting that the decline in T3 may be an adaptive response for energy preservation (232,233). This adaptive decline in T3 may be mediated, in part, by the fall in leptin levels that accompanies weight loss as it can be reversed with leptin administration (234). Subcutaneous and visceral fat showed reduced thyroid gene expression in subjects with obesity, especially TSH Receptor gene expression. These changes were reversed by major weight loss (235).

After weight loss from bariatric surgery, FT3 and TSH levels were significantly reduced and serum thyroperoxidase antibody (TPOAb) and thyroglobulin antibody (TgAb) levels decreased significantly from 79.3 and 177.1 IU/mL to 57.8 and 66.0 IU/mL, respectively, in participants with positive thyroid antibodies (236). Also in patients starting with a subclinical hypothyroidism state, weight loss leaded to normalization of TSH levels in most patients and none developed overt hypothyroidism (237). Furthermore, in children with obesity and overweight without circulating antithyroid antibodies, BMI reductions uniquely predict reductions in TSH, thyroid volume, and improvement in thyroid structure with an altered parenchymal pattern at thyroid ultrasound (238).

Body mass index is directly associated with thyroid volume and the incidence of thyroid nodules. This association appears to be in positive correlation with the degree of insulin resistance (221,239,240). Not only is the incidence of benign thyroid abnormalities increased in obesity, but a higher rate of malignancy has also been reported (241,242). Pathway analysis has identified 1,036 genes associated with thyroid cancer (TC) and 534 regulated by obesity. Five out of the 358 obesity-specific genes, FABP4, CFD, GHR, TNFRSF11B, and LTF, had significantly decreased expression in TC patients (243). Hyperinsulinemia is a common factor found in most studies linking obesity with increased thyroid cancer incidence (244,245). It is not surprising that particularly high percentage of visceral fat mass has a stronger association with thyroid cancer since VAT is highly metabolically active and associated with increased IR. Even though neck circumference as an index of upper-body adiposity, had a positive correlation with thyroid cancer tumor size and lymph node metastasis (246), other studies do not observe any association between obesity and thyroid cancer aggressive features (247,248). Whether obesity increases the risk of thyroid cancer remains controversial as several authors have concluded that obesity is associated with greater risk of thyroid cancer (249,250), while others do not (251).

Synthetic thyroid hormones, as well as various other thyroid hormone preparations, have been used as adjunctive measures to induce or facilitate weight loss. A systematic review reported by Kaptein el al (252) recognized 14 randomized controlled trials and prospective observational studies describing the effects of T3 and T4 therapy in comparison with placebo in euthyroid subjects with obesity during caloric deprivation. Most of these studies had a small sample size, ranging from 5 to 12 treated patients. Thyroid hormone treatment resulted in subclinical hyperthyroidism in most patients and there was no consistent effect on weight loss across the studies.

Since the action of thyroid hormone varies depending on the activated receptor, selective thyroid receptor agonists have been developed. In brief, thyroid hormones exert their actions through two major receptors: thyroid receptor alpha (TRA), which mainly mediates T4 effects in bone, skeletal muscle, brain and heart, and thyroid receptor beta (TRB) that regulates TRH/TSH secretion and the metabolic effects of T3 in the liver, such as lowering lipids.

Adipose tissue expresses both TRs (253). Selective TRB agonists are promising drugs for treatment of dyslipidemia and obesity without the toxic effects of thyroid hormones analogs on bones or heart in euthyroid patients. This has been tested in animal studies but there are no clinical trials in humans yet (254-256).

CONCLUSIONS AND CLINICAL IMPLICATIONS

As discussed in the previous sections, several endocrine alterations can be identified in association with obesity (Table 4). In most cases, these alterations are reversible with weight loss and, therefore, appear to be a consequence of obesity. Emphasis has been focused on the hypothalamic-pituitary hormones axes and the possibility that some "subclinical" alterations in these axes may be at the origin of increased adiposity. At this time this hypothesis needs further testing. What is true is that the interaction between the adipose tissue and the body is far more complex than once believed, and the future will certainly provide more decisive data on the precise mechanisms of these interactions and their contribution to the development and/or the maintenance of obesity.

Certain endocrine syndromes are known to result in obesity. From the clinical practitioner's perspective it is important to remember these syndromes and to be suspicious should a patient with obesity display one or more of the clinical features seen in these disorders. Hypothyroidism is a common clinical problem and can, of course, occur in patients with obesity and could contribute to the presence of symptoms such as fatigue and inability to concentrate. Hypothyroidism is under-diagnosed in the general population and specifically patients with obesity. Routine screening of patients who present with obesity with a sensitive TSH assay and free T4 is reasonable, although there are no specific guidelines with regards to this. Cushing's syndrome is frequently included in the differential diagnosis of obesity and patients with abdominal obesity have many features in common with patients with authentic Cushing's. However, true Cushing's disease (due to excessive endogenous corticosteroids) is rare. Nevertheless, if there is a reasonable suspicion for this condition, the patient should be screened. Attention should be focused on symptoms and signs that are more specific to Cushing's such as proximal muscle weakness, purple striae, thin and bruised skin, hypokalemia, and osteopenia.

Hypogonadism and growth hormone deficiencies are both associated with abdominal obesity. The former is very common and should be kept in mind in males with other symptoms or signs suggestive of androgen deficiency while the latter is usually suspected in the setting of surgery or disease of the hypothalamus-pituitary axis. The treatment of these two conditions can result directly and indirectly (by improving conditioning, muscle strength, and stamina) in weight loss, improved metabolic profile, and improved bone density but is usually reserved for those with true deficiencies, not with low-normal levels.

Table 4. Hormonal Changes in Obesity
Adipose tissue as an endocrine organ
	Type 2 obesity with leptin resistance  Leptin increases
	Type 1 obesity with congenital leptin deficiency  Leptin decreases
	Adiponectin decreases
	Chemerin increases
	Omentin decreases
	Retinol Binding Protein increases
	Angiotensin 2 increases
	Plasminogen activator inhibitor-1 (PAI-1) increases
	Interleukin-6 increases
Obesity and the pituitary axes
	LH pulsatility decreases
	Total testosterone decreases in men
	Free testosterone decreases in men
	SHBG decreases in women and men
	Androgens increase in women
	Free testosterone increases in women
	Androstenedione decreases in women
	Increase in kisspeptin levels
	Aromatization of androgens in adipocytes leads to elevated estrogens levels
	GH level decreases
	GH binding protein increases
	IGF-1 normal or slightly low
	Cortisol normal
	24-hour urine free cortisol (UFC) excretion high-normal
	TSH normal or slightly increased

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46,XY Differences of Sexual Development

ABSTRACT

The 46,XY differences of sex development (46,XY DSD) can result either from decreased synthesis of testosterone and/or DHT or from impairment of androgen action. 46,XY DSD are characterized by micropenis, atypical or female external genitalia, caused by incomplete intrauterine masculinization with or without the presence of Müllerian structures. Male gonads are identified in the majority of 46,XY DSD patients, but in some of them no gonadal tissue is found. Complete absence of virilization results in normal female external genitalia and these patients generally seek medical attention at pubertal age, due to the absence of breast development and/or primary amenorrhea. A careful clinical evaluation of the neonate is essential because most DSD patients could be recognized in this period and prompt diagnosis allows a better therapeutic approach. Family and prenatal history, complete physical examination and assessment of genital anatomy are the first steps for a correct diagnosis. The diagnostic evaluation of DSD includes hormone measurements (assessment of Leydig and Sertoli cell function), imaging (ultrasonography is always the first and often the most valuable imaging modality in DSD patients’ investigation), cytogenetic, and molecular studies. Endoscopic and laparoscopic exploitation and/or gonadal biopsy are required in very few cases. Psychological evaluation is of crucial importance to treat DSD patients. Every couple that has a child with atypical genitalia must be assessed and receive counseling by an experienced psychologist, specialized in gender identity, who must act as soon as the diagnosis is suspected, and then follow the family periodically, more frequently during the periods before and after genitoplasty. Parents must be well informed by the physician and psychologist about normal sexual development. A simple, detailed, and comprehensive explanation about what to expect regarding integration in social life, sexual activity, need of hormonal and surgical treatment and the likely possibility or not of fertility according to the sex of rearing, should also be discussed with the parents before the assignment of final social sex. Optimal care of DSD patients begins in the newborn period and sometimes in prenatal life and requires a multidisciplinary team. Most of the well-treated DSD patients present a normal quality of life in adulthood.

INTRODUCTION

Male phenotypic development is a 2-step process: 1) testis formation from the primitive gonad (sexual determination) and 2) internal and external genitalia differentiation by action of factors secreted by the fetal testis (sexual differentiation). The first step is very complex and involves interplay of several transcription factors and signaling cells (1-3). Dosage imbalances in genes involved in DSD (deletions or duplication) have also been identified as a cause of these developmental differences (Fig. 1).

Figure 1. Summary of the molecular events in sex determination indicating the genes in which molecular defects can cause gonadal disorders in animal models. Some of these disorders were confirmed in humans. Nr5a1, Wnt4 and Wt1 are expressed in the urogenital ridge whose development results in formation of the gonads, kidneys, and adrenal cortex. Several genes, Wt1, Nr5a1, M33 (CBX2 mouse homologue), Lhx9, Lim1, Gata4/Fog2, Dmrt1, Emx2 and Cited are expressed in the bipotential gonad. Nr5a1 up-regulates Cbx2 expression that is required for upregulation of the Sry gene. Nr5a1 and Wt1 up-regulate Sry expression in pre-Sertoli cells and Sry initiates male gonad development. Sry strongly up-regulates Sox9 in Sertoli cells. Sox9 up-regulates Fgf9 and Fgf9 maintains Sox9 expression, forming a positive feed-forward loop in XY gonads. The balance between Fgf9 and Rspo1/Wnt4 signals is shifted in favor of Fgf9, establishing the male pathway. If Wnt4/Rspo1 is overexpressed activating the β-catenin pathway, this system blocks Fgf9 and disrupts the feed-forward loop between Sox9 and Fgf9. Pdg2 signaling up-regulates Sox9 and Sox9 activate Ptgds. Sox9 establishes a feed-forward loop with the Pgd2. Sox9 inhibits beta-catenin-mediated Wnt signaling. Overexpression in either Dax1 (locus DSS) or Rspo1/Wnt4 antagonizes testis formation. On the other hand, Dax1 regulates the development of peritubular myoid cells and the formation of testicular cords. Dmrt1 has recently been shown to be required for the maintenance of gonadal sex and to prevent female reprogramming in postnatal testis, Cbx2 directly or indirectly repress ovarian development. Dhx37 has critical roles in early human testis determination and also in the maintenance of testicular tissue.

The second step, male sex differentiation, is a more straightforward process. Mesonephric (Wolffian) and paramesonephric (Mullerian) ducts are present in both, male and female fetuses, and originate from the anterolateral epithelium of the urogenital ridge. Anti Müllerian hormone (AMH) secreted by the testicular Sertoli cells acts on its receptor in the Müllerian ducts to cause their regression. Testosterone secreted by the testicular Leydig cells acts on the androgen receptor in the Wolffian ducts to induce the formation of epididymis, deferent ducts and seminal vesicles (Fig. 2) (4). The external genitalia of the fetus derive from mesenchyme cells from the primitive streak. Under androgen stimuli male fetal urethral folds, genital tubercle and genital swellings give rise to corpus spongiosum and primitive urethra, phallus, and scrotal swellings respectively. This process is mediated by testosterone and its further reduced dihydrotestosterone (DHT), which acts on the androgen receptor of the prostate and external genitalia leading to their masculinization (5,6) (Figs. 3 - 8).

Figure 2. Summary of the molecular events in sex differentiation indicating the genes in which molecular defects cause 46,XY DSD in humans. After testis determination, hormones produced by the male gonad induce the differentiation of internal and external genitalia acting on their specific receptor. The regulation of AMH gene requires cooperative interaction between SOX9 and NR5A1, WT1, GATA4 and HSP70 at the AMH promoter. Combined expression of DHH, MAMLD1 and NR5A1 is required for Leydig cell development. NR5A1 regulates gonadal steroidogenesis. The Leydig cells also produce INSL3, which causes the testes to descend to the scrotum.

Figure 3. The development of male internal genitalia in the human embryo. The 6-wk-end embryo is equipped with both male and female genital ducts derived from the mesonephrons.

Figure 4. The development of male internal genitalia in the human embryo. The regression of the Müllerian ducts is mediated by the action of AMH secreted by the fetal Sertoli cells.

Figure 5. The development of male internal genitalia in the human embryo. The stabilization and differentiation of the Wolffian ducts are mediated by testosterone synthesized by the fetal Leydig cells. The enzyme 5α-reductase 2 converts testosterone to dihydrotestosterone (DHT). The Wolffian ducts differentiate into epididymis, vas deferens and seminal vesicles. DHT contributes to prostate differentiation.

Figure 6. Development of male external genitalia in the human embryo. At the 8-wk-end embryo the external genitalia of both sexes are identical and have the capacity to differentiate in both directions: male or female. DHT stimulates growth of the genital tubercle and induces fusion of urethral folds and labioscrotal swellings. It also inhibits growth of the vesicovaginal septum, preventing development of the vagina.

Figure 7. Development of male external genitalia in the human embryo. At the 12-week-end embryo the male external genitalia are entirely formed.

Figure 8. Development of male internal and external genitalia in the human embryo. At the 12-week-end embryo both internal and external genitalia are completely formed.

The term differences of sex development (DSD) include- congenital conditions in which development of chromosomal, or gonadal or anatomical sex is atypical. This nomenclature has been proposed to replace terms such as intersex, pseudo-hermaphroditism and sex reversal (6,7). These terms, previously used to describe the differences of sex development, are potentially offensive to the patients and the consensus on the management of intersex disorders recommends a new nomenclature that will be followed in this chapter (6). The proposed changes in terminology aim to integrate upcoming advances in molecular genetics in the most recent DSD classification (8).

The 46,XY DSDs are characterized by micro-penis, atypical or female external genitalia, caused by incomplete intrauterine masculinization with or without the presence of Müllerian structures. Male gonads are identified in the majority of 46,XY DSD patients, but in some of them no gonadal tissue is found. Complete absence of virilization results in normal female external genitalia and these patients generally seek medical attention at pubertal age, due to the absence of breast development and/or primary amenorrhea. 46,XY DSD can result either from decreased synthesis of testosterone or DHT or from impairment of androgen action (9,10). Our proposal classification of 46,XY DSD is displayed in Table 1.

Table 1. Classification of 46,XY DSD

46,XY DSD DUE TO ABNORMALITIES OF GONADAL DEVELOPMENT

Gonadal agenesis

Gonadal dysgenesis - complete and partial forms

46,XY DSD ASSOCIATED WITH CHOLESTEROL SYNTHESIS DEFECTS

Smith-Lemli-Opitz syndrome

46,XY DSD DUE TO TESTOSTERONE PRODUCTION DEFECTS

Impaired Leydig cell differentiation (LHCGR defects)

Complete and partial forms

Enzymatic defects in testosterone synthesis

Defects in adrenal and testicular steroidogenesis

STAR deficiency

P450scc deficiency

3-β-hydroxysteroid dehydrogenase II deficiency

17α-hydroxylase and 17,20 lyase deficiency

P450 oxidoreductase defect (electron transfer disruption)

Defects in testicular steroidogenesis

Isolated 17,20-lyase deficiency

Cytochrome b5 defect (allosteric factor for P450c17 and POR interaction)

17β-hydroxysteroid dehydrogenase III deficiency

Alternative pathway to DHT

3α- hydroxysteroid dehydrogenase deficiency due to AKR1C2 and AKR1C4 defects

46,XY DSD DUE TO DEFECTS IN TESTOSTERONE METABOLISM

5α-reductase type 2 deficiency

46,XY DSD DUE TO DEFECTS IN ANDROGEN ACTION

Androgen insensitivity syndrome

Complete and partial forms

46,XY DSD DUE TO PERSISTENCE OF MÜLLERIAN DUCTS

Defect in AMH synthesis

Defect in AMH receptor

CONGENITAL NON-GENETIC 46,XY DSD

Maternal intake of endocrine disruptors

Associated with impaired prenatal growth

46,XY OVOTESTICULAR DSD

NON-CLASSIFIED FORMS

Hypospadias

46,XY gender dysphoria

INVESTIGATION OF DSD PATIENTS

Optimal care of patients with DSD requires a multidisciplinary team and begins in the newborn period. A careful clinical evaluation of the neonate is essential because most DSD patients could be recognized in this period and prompt diagnosis allows a better therapeutic approach. Family and prenatal history, complete physical examination and assessment of genital anatomy are the first steps for a correct diagnosis. The diagnostic evaluation of DSD includes hormone measurements, imaging, cytogenetic, and molecular studies (11). In very few cases, endoscopic and laparoscopic exploitation and/or gonadal biopsy are required (12).

The endocrinological evaluation of 46,XY DSD infants includes assessment of testicular function by basal measurements of LH, FSH, inhibin B, anti-Mullerian hormone (AMH), and steroids. AMH and inhibin B are useful markers of the presence of Sertoli cells and their assessment could help in the diagnosis of testis determination disorders. In boys with bilateral cryptorchidism serum AMH and inhibin B correlate with the presence of testicular tissue and undetectable values are highly suggestive of absence of testicular tissue (13,14).

In minipuberty and in postpubertal patients with testosterone synthesis defects, the diagnosis is made through basal steroid levels. Testosterone levels are low and steroids upstream from the enzymatic blockage are elevated. This pattern can be confirmed by an hCG stimulation test, which increases the accumulation of steroids before the enzymatic blockage, with a slight elevation of testosterone. In prepubertal individuals, an hCG stimulation test is essential for the diagnosis, since basal levels are not altered.

There are several hCG stimulation protocols and normative data must be established for each of them. We established a normal testosterone response 72 and 96 hours after the last of 4 doses of hCG, 50-100 U/kg body weight, given via intramuscular every 4 days in boys with cryptorchidism but otherwise normal external genitalia: testosterone peak levels reached 391 ± 129 ng/dL and we consider a subnormal response a value <130 ng/dL (equivalent to -2 SD) (15).

Imaging evaluation is indicated in the neonatal period when atypical genitalia are identified. If apparent female genitalia with clitoral hypertrophy, posterior labial fusion, foreshortened vulva with single opening or inguinal/labial mass is present, imaging studies may also be performed. A family history of DSD and later presentations as abnormal puberty or primary amenorrhea, cyclic hematuria in a male, and inguinal hernia in a female also require an imaging evaluation.

Ultrasonography is always the first and often the most valuable imaging modality in DSD patients’ investigation. Ultrasound shows the presence or absence of Müllerian structures at all ages and can locate the gonads and characterize their echo texture. This exam can also identify associated malformations such as kidney abnormalities (16).

Genitography and cystourethrography can display the type of urethra, the presence of vagina, cervix, and urogenital sinus. MRI contributes to accurate morphologic evaluation of Mullerian duct structures, the gonads, and the development of the phallus, all of which are essential for appropriate gender assignment and planning of surgical reconstruction (17).

CYTOGENETIC AND MOLECULAR INVESTIGATION

The routine use of genetic testing for reaching a diagnosis in XY DSD is increasingly playing an important role in the diagnostic process. A wide range of techniques may be used, each one having a different investigative application and genetic resolution (18,19).

More than 75 genes involved in gonadal development and/or sex hormone biosynthesis/action are known causes of DSDs and the molecular methodologies have contributed to identify already known as well as novel causes of DSD. These results have led to the adoption of molecular tests into clinical practice for diagnosis and genetic counseling, reducing the need of hormonal and imaging tests to reach the correct diagnosis (20). Advances in molecular biological techniques for diagnosing DSD are reviewed in recent publications (18,19).

Chromosomal Analysis

Early identification of chromosomal regions and candidate-genes involved in the DSD etiology

were established by finding microscopically visible structural changes in the karyotype, using conventional cytogenetic techniques. Many of them were achieved by positional cloning and linkage analysis, which are not widely used tools.

Although conventional karyotyping is still used frequently in routine clinical diagnosis, faster molecular cytogenetic techniques that do not require cell culture can be employed. Array techniques [array comparative genomic hybridization (aCGH) and single-nucleotide polymorphism (SNP) array] are all capable to identify submicroscopic genome imbalance / copy number variation (CNV), as small as 10 KB (CNVs between 10 kb and 5 Mb in size), and which may affect several genes, in patients with an apparently normal karyotype (21,22).

CNV affecting coding sequences or regulatory elements of critical dosage-sensitive genes are known causes of DSDs (23-26). Novel DSD candidate chromosomic regions and genes, with potential roles in sex determination and DSD, such as SUPT3H, C2ORF80, KANK1, ADCY2, VAMP7 and ZEB2, have been also identified by array studies, many of them waiting for further validation (25,27).

Array techniques can diagnose pathogenic CNV in almost 30% of syndromic DSD patients as a single method (27,28). Thus, a CGH or SNP-array was proposed as the first genomic test to investigate this group of DSD patients.

Sequencing Analysis

Among the genetic tests, many use a candidate-gene approach (Sanger sequencing), while targeted DSD gene panels, wider whole-exome (WES) and whole-genome (WGS) scale are high-throughput screening technologies, in which multiple short DNA target sequences are analyzed to identified the presence of allelic variants (29).

Sanger sequencing is often the method of choice if a specific genetic condition is highly suspected by an established clinical and hormonal diagnosis. AR and SRD5A2, in addition to almost all testosterone synthesis defects, are the most requested genes in 46,XY DSD to be sequenced using this approach (20).

The superiority of targeted DSD gene panel tests, that can evaluate simultaneously several and non-standard sets of genes, over single-gene testing approaches is well established, especially considering time and cost-effectiveness (26,30).

Whole-Exome Sequencing (WES) and Whole-Genome Sequencing (WGS) are also based on short-read sequencing. They present a clear improvement over single-gene testing in providing clinical diagnosis for DSD. The advantage of WES/WGS is the potential to identify new DSD-related genes in the research setting. On the other hand, WGS has more consistent coverage of gene sequences throughout the genome, including the non-coding regions and so it has the potential to provide a much higher diagnostic yield than WES (25,31).

Nevertheless, WES and WGS require significant bioinformatic resources and are expensive strategies; consequently, their application for first-line diagnostic investigation in many clinical settings are still limited (18,32).

The target DNA can also be read in longer fragments (several kb). The main advantage of using long-reads during the process is that repetitive elements and complex structural variants can often be resolved to a greater extent than in assemblies generated from short-read sequencing (18). Long-read sequencing offers a potential solution to genome-wide short tandem repeats analysis, which are highly variable elements, which play a pivotal role in the regulation of gene expression.

Studies in animal models have suggested the involvement of epigenetic regulation in the process of gonadal formation, reinforcing a probable role of epigenetic variation in the etiology of DSD (33).

Careful selection of the genetic test indicated for each condition remains important for a good clinical practice (Figure 9).

Figure 9. Algorithm for 46,XY DSD diagnosis

46,XY DSD DUE TO ABNORMALITIES IN GONADAL DEVELOPMENT

Uncountable allelic variants identified in several genes involved in the process of human gonadal determination have been associated with 46,XY gonadal dysgenesis. They will be described according to the period of gene expression in gonadal determination.

Gonadal Determination and Differentiation

The intermediate mesoderm is the primary embryonic tissue at gastrulation that gives rise to the urogenital ridge. This, in turns, is going to derive the primitive gonad from a condensation of the medioventral region of the urogenital ridge. The primitive gonad separates from the adrenal primordium at about 5 weeks but remains bipotential until the 6^thweek after conception. Mammals sex determination is a complex process, which involves many genes acting in networks. Several genes have been involved in the development of the urogenital ridge, including Emx2, Lim1, Lhx9, Wt1, Gata-4/Fog2, Nr5a1/Sf1. Although knockout models of these genes produce abnormal gonads in mice, not all of them have been implicated in the human gonadal dysgenesis etiology.

To date, Emx2 null mice have absent kidneys, ureters, gonads and genital tracts and have developmental abnormalities of the brain (34). In humans, variants in EMX2 have been found in patients with schizencephaly (a rare condition in which a person is born with clefts in the brain that are filled with liquor) but no gonadal phenotypes have been described. WT1, NR5A1 and NR0B1/DAX1 are well known genes that are critical for the formation of the urogenital ridge in humans. The products of WT1 are essential for both gonadal and renal formation (35) whereas NR5A1/SF1 protein is essential for gonadal and adrenal formation (36,37). NR0B1/DAX1 is also essential for gonadal and adrenal differentiation and when mutated, results in congenital adrenal hypoplasia and hypogonadotropic hypogonadism (38).

After the formation of the bipotential gonad, by the 6^th week after conception, in 46, XY individuals, the expression of the testis-determining gene Sry, which is transcriptionally regulated by the expression of Wt1 (39) and GATA Binding Protein 4 (Gata4), its cofactor the Friend-of-GATA (Fog2) (40) and chromobox protein homolog 2 (Cbx2) (41) trigger the gonadal masculinizing fate process. In the mammalian male embryo, the first molecular signal of sex determination is the expression of Sry within a subpopulation of somatic cells of the indifferent genital ridge (42). The transient expression of Sry drives the initial differentiation of pre-Sertoli cells that would otherwise follow a female pathway, becoming granulosa cells. Once Sry expression begins, it initiates the cascade of gene interactions and cellular events that direct the formation of a testis from the undifferentiated fetal gonad. So, pre-Sertoli cells proliferate, polarize and aggregate around the germ cells to define the testes cords. Migration of cells into the gonad from the mesonephros or the coelomic epithelium is subsequently induced by signals emanating from the pre-Sertoli cells. Peritubular myoid cells surround the testes cords and cooperate with pre-Sertoli cells to deposit the basal lamina and further define the testis cords. Signaling molecules produced by the pre-Sertoli cells promote the differentiation of somatic cells, found outside the cords, into fetal Leydig cells, thus ultimately allowing the production of testosterone. Endothelial cells are associated to form the coelomic vessel, which promotes efficient export of testosterone into plasma.

The gene Sox9 is up-regulated immediately after Sry expression and is involved in the initiation and maintenance of Sertoli cell differentiation during the early phases of testis differentiation. The mechanism by which NR5A1 and SRY increase endogenous SOX9 expression was clearly demonstrated in human embryonal carcinoma cell line NT2/D1 (43).

Extracellular signaling pathways (Fgf9 and Igf1r/Irr/Ir) play a significant role in Sox9 expression. A model has been suggested in that the fate of the bipotential gonad is controlled by mutually antagonistic signals between Fgf9 andWnt4/Rspo1. In this model Sox9 up-regulates Fgf9-Fgfr2 and Fgf9 maintains Sox9 expression, forming a positive feed-forward loop in XY gonads. The balance between Fgf9 and Wnt4/Rspo1 signals is shifted in favor of Fgf9, establishing the male pathway. In addition, Sry inhibits β-catenin-mediated Wnt signaling (44). In the absence of this feed-forward loop between Sox9 and Fgf9, Wnt4/Rspo1, the activated β-catenin pathway, blocks Fgf9 and promotes the ovarian fate (45,46). Furthermore, Sox9 directly binds to the promoter of the Ptgds gene which encodes prostaglandin D synthase that mediates the production of PGD2 (47) which, in turn, promotes nuclear translocation of Sox9, facilitating Sertoli cell differentiation (48). Antagonism between Dmrt1 and Foxl2 comprises another step for sex-determining decision. Dmrt1 has been described as essential to maintain mammalian testis determination, preventing female reprogramming in the postnatal mammalian testis (49). MAP3K1 has been described to be important to the balance between SOX9/FGF9 to WNT/beta-catenin signaling in functional studies (50,51). However, the role of MAP3K1 in human sex-determination remains unknown as the downstream effectors of MAP3K1 in the human developing testis have not been identified (52). Similarly, the precise mechanism by which DHX37 interferes with testis determination/maintenance remains to be elucidated (53). Abnormalities in the expression (underexpression or overexpression or timing of expression) of genes involved in the cascade of testis determination can cause anomalies of gonadal development and consequently, 46,XY DSD. The absence, regression, or the presence of dysgenetic testes results in abnormal development of the genital ducts and/or external genitalia in thosepatients.

46,XY Gonadal Agenesis

Total absence of gonadal tissue confirmed by laparoscopy has rarely been described in XY subjects with female external and internal genitalia indicating the absence of testicular determination (54). Mendonca et al described a pair of siblings, one XY and the other XX, born to a consanguineous marriage, with normal female external and internal genitalia associated with gonadal agenesis (55). Pathogenic allelic variants in NR5A1 and LHX9 were later ruled out in these siblings (56). The origin of this disorder remains to be determined, but a defect in another gene essential for bipotential gonad development is the most likely cause of this disorder.

46,XY Gonadal Dysgenesis - Complete and Partial Forms

46,XY gonadal dysgenesis consists of a variety of clinical conditions, in which the development of the fetal gonad is abnormal and encompasses both a complete and a partial form. The complete form of gonadal dysgenesis was first described by Swyer et al. (57) and is characterized by female external and internal genitalia, lack of secondary sexual characteristics, normal or tall stature without somatic stigmata of Turner syndrome, eunuchoid habitus and the presence of bilateral dysgenetic gonads in XY subjects. Mild clitoromegaly is present in some cases.

The partial form of this syndrome is characterized by variable degrees of impaired testicular development and testicular function. These patients present a spectrum of atypical genitalia with or without Müllerian structures. Similar phenotypes can also result from a 45,X/46,XY karyotype.

Serum gonadotropin levels are elevated in both the complete and partial forms, mainly FSH levels, which predominate over LH levels. Testosterone levels are at the prepubertal range in the complete form. Meanwhile, in the partial form, it can range from prepubertal levels to normal adult male levels.

The clinical condition named embryonic testicular regression syndrome (ETRS) has been considered part of the clinical spectrum of partial 46,XY gonadal dysgenesis (58). In this syndrome, most of the patients present atypical genitalia or micropenis associated with complete regression of testicular tissue in one or both sides. Pathogenic/likely pathogenic variants in DHX37 were reported in patients with 46,XY GD at a frequency of 14%. Considering only the ETRS phenotype (micropenis and absence of uni- or bilateral testicular tissue), this frequency increases to 50% (53).The masculinization degrees of internal and external genitalia presented are related to the time and duration of the hormonal secretion, prior to cessation of testicular function. The dysgenetic testes showed disorganized seminiferous tubules and stroma with occasional primitive sex cords without germ cells (59). Familial cases of gonadal dysgenesis with variable degrees of genital atypia have been reported, and the nature of the underlying genetic defect is still unknown in several families, despite new genetic investigation methodologies available (58). Regarding the genetic etiology, 46,XY gonadal dysgenesis is heterogeneous and can result from defects of any gene involved in the process of gonadal formation.

The following review will focus on the main genes causing gonadal dysgenesis in humans, presenting as an isolated or syndromic phenotype.

Dysgenetic 46,XY DSD Due to Under Expression of GATA4 and FOG2/ ZFPM2 Genes

Gata4 (GATA-binding factor 4 gene) cooperatively interacts with several proteins to regulate the expression of genes involved in testis determination and differentiation as SRY, SOX9, NR5A1, AMH, DMRT1, STAR, CYP19A1, and others (60).

In humans, GATA4 variants were first described in patients with congenital heart defects without genital abnormalities (61). However, genitourinary anomalies, such as hypospadias and cryptorchidism, were described in 46,XY patients with deletion of the 8p23.1 region, in which GATA4 is located (62).

The p.G221R GATA4 pathogenic variant was identified in five members of a French family, three 46,XY DSD patients, two of them with cardiac anomalies, and in their apparently unaffected mothers (63).

The role of FOG2 in human testis development was corroborated by the identification of a balanced translocation (8;10) (q23.1;q21.1) in a patient with partial gonadal dysgenesis and congenital heart abnormalities (64). Bashamboo et al. identified missense FOG2 variants, using exome sequencing, in two patients with 46,XY gonadal dysgenesis. One patient carried the non-synonymous p.S402R heterozygous variant. The second patient carried the inherited homozygous p.M544I variant and the de novo heterozygous p.R260Q variant. The p.M544I variant by itself has little effect on the biological activity of FOG2 protein in transactivation of the gonadal promoters, but it shows reduced binding with GATA4. In the in vitro assays, a combination of both the p.R260Q and the p.M544I variants altered the biological activity of the FOG2 protein on specific downstream targets, as well as obliterated its interaction with GATA4. In the patient, the two variants together may result in an imbalance of the delicate equilibrium between antagonistic male and female pathways leading ultimately to gonadal dysgenesis (65). Although several GATA4 and FOG2/ZFPM2 variants have been identified in 46,XY DSD patients, the real role of the majority of them in the etiology of gonadal disease is still unclear. The re-study of seven GATA4 and ten FOG2/ZFPM2 variants previously identified by Eggers et al. (26) in a cohort of 46,XY DSD patients, using updated tools and testing their molecular activity in the context of gonadal signaling by in vitro assays, support that the majority of them are benign in their contribution to 46,XY DSD. Only one variant (p. W228C) located in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH, and in protein interaction with ZFPM2 (66).

Dysgenetic 46,XY DSD Due to Under Expression of the CBX2 gene

In humans, variants in both CBX2.1 and CBX2.2 isoforms were associated with 46,XY DSD (67,68).

The compound heterozygous CBX2.1 variants, c.C293T (p.P98L) and c.G1370C (p.R443P), inherited from the father and the mother respectively, was identified in a 46,XY patient, who was born with a completely normal female phenotype. The patient had uterus and histologically normal ovaries (67) and high serum FSH levels. Her phenotype resembles the Cbx2 knock-out XY mice phenotype (41). Cbx2 (M33) knockout mice present hypoplastic gonads in both sexes, but a small or absent ovaries are observed in the XY Cbx2 knockout, consequently to the reduced expression of Sry and Sox9 in the gonadal tissue (41). Functional studies demonstrated that these variants do not bind to, or adequately regulate the expression of target genes important for gonadal development, such as NR5A1 (67).

Mutated CBX2.2 isoforms were also implicated in the etiology of partial 46,XY gonadal dysgenesis in two other patients. Each patient carried a distinct variant, the p. C132R (c.394T>C) and the C154fs (c.460delT). These CBX2.2variants were shown to be related to a defective expression of EMX2 in the developing gonad (68). However, analysis of populational genetics data indicates that p.C154fs is present in general populations at high frequency, inconsistent with causing gonadal dysgenesis (69).

46,XY DSD Due to Under Expression of the WT1 Gene

The Wilms’ tumor suppressor gene (WT1) is located on 11p13, and encodes a zinc-finger transcription factor involved in the development and function of the kidneys and gonads. The WT1 contains 10 exons, of which exons 1–6 encode a proline/glutamine-rich transcriptional-regulation region and exons 7–10 encode the four zinc fingers of the DNA-binding domain. Four major species of RNA with conserved relative amounts, different binding specificities, and different subnuclear localizations are generated by two alternative splicing regions (70). Splicing at the first site results in either inclusion or exclusion of exon 5. The second alternative splicing site is in the 3’ end of exon 9 and allows the inclusion or exclusion of three amino acids lysine, threonine and serine between the third and fourth zinc fingers, resulting in either KTS-positive or negative isoforms. Isoforms that only differ by the presence or absence of the KTS amino acids have different affinities for DNA and, therefore, possibly different regulatory functions (70). A precise balance between WT1 isoforms is necessary for its normal function (71).

WT1 presents a complex network of interaction with several protein systems so that abnormalities in it can determine a wide phenotypic spectrum in XY and XX individuals (72). Several syndromes are associated with WT1 pathogenic variants, including: WAGR, Denys-Drash and Frasier syndrome.

WAGR syndrome is characterized by Wilms’ tumor, aniridia, genitourinary abnormalities and mental retardation. Genitourinary anomalies are frequently observed, such as renal agenesis or horseshoe kidney, urethral atresia, hypospadias, cryptorchidism and more rarely atypical genitalia (73). Heterozygous deletions of WT1 and othercontiguous genes are the cause of this syndrome (74). Deletions of PAX6 gene are related to the presence of aniridia in these patients. Severe obesity is present in a substantial proportion of subjects with the WAGR syndrome, and the acronym WAGRO has been suggested for this condition (75). The phenotype of obesity and hyperphagia in WAGRO syndrome is attributable to deletions that determine haploinsufficiency of the BDNF gene (Brain-Derived Neurotrophic Factor) (76).

Le Caignec et al. described a 46,XY patient with an interstitial deletion of approximately 10 Mb located on 11p13, encompassing WT1 and PAX6 , who presented a female external and internal genitalia, an unusual phenotype of WAGR syndrome (77). This report demonstrated an overlap of clinical and molecular features in WAGR, Frasier and Denys-Drash syndromes that confirms these conditions as a spectrum of disease due to WT1 alterations.

Denys-Drash syndrome is characterized by dysgenetic 46,XY DSD associated with early-onset renal failure (steroid-resistant nephrotic syndrome with diffuse mesangial sclerosis and progression to end-stage kidney disease) and Wilms´ tumor development in the first decade of life (78). Müllerian duct differentiation varies according to the Sertoli cells' function. The molecular defect of this syndrome is the presence of heterozygous missense allelic variants in the zinc finger encoding exons (DNA-binding domain) of WT1 gene (79). Gonadal development is impaired to variable degrees, resulting in a spectrum of 46,XY DSD (80).

Frasier syndrome is characterized by a female to atypical external genitalia phenotype in 46,XY patients, streak or dysgenetic gonads, which are at high risk for gonadoblastoma development, and renal failure (early steroid-resistant nephrotic syndrome with focal and segmental glomerular sclerosis). In these patients the progression to end-stage renal disease often occurs in adolescence, although the late-onset nephropathy has also been described in Frasier syndrome (81), reinforcing that patients carrying WT1 pathogenic variants should have the renal function carefully monitored (82).

Constitutional heterozygous variants of the WT1, almost all located at intron 9, are found in patients with Frasier syndrome, leading to a change in splicing that results in reversal of the normal KTS positive/negative ratio from 2:1 to 1:2 (78,83). Exonic variants have been also associated with Frasier syndrome (84).

The report of atypical external genitalia (84) , the presence of Wilms’ tumor (85), and the description of exonic variants in the DNA binding domain of WT1 gene (84) in patients with Frasier syndrome indicate an overlap of clinical and molecular features between Denys Drash and Frasier syndromes.

46,XY DSD Due to Under Expression of the NR5A1/SF1 Gene

NR5A1 was originally identified as a master-regulator of steroidogenic enzymes in the early 1990s following the Keith L. Parker and Kenichirou Morohashi inspiring work (86-88). NR5A1 has since been shown to control many aspects of adrenal and gonadal function (37,89), NR5A1, together with several signaling molecules, are also involved in adrenal stem cell maintenance, proliferation and differentiation inducing adrenal zonation, probably acting in the progenitor cells (90).

Homozygous 46,XY null mice (−/−) have adrenal agenesis, complete testicular dysgenesis, persistent Müllerian structures, partial hypogonadotropic hypogonadism, and other features such as late-onset obesity (91). Therefore, it was clear demonstrated that Nr5a1 is an essential factor in sexual and adrenal differentiation, and a key regulator of adrenal and gonadal steroidogenesis and also of the hypothalamic-pituitary-gonadal axis.

The first reported human case of NR5A1 pathogenic variant, the heterozygous p.G35E, was a 46,XY patient who presented female external genitalia and Müllerian duct derivatives, indicating the absence of male gonadal development, associated with adrenal insufficiency. This patient presented with salt-losing adrenal failure in early infancy and was thought to have a high block in steroidogenesis (e.g., in CYP11A1, STAR) affecting both adrenal and testicular functions. However, the identification of streak-like gonad and Müllerian structures was consistent with testicular dysgenesis, thereby, a disruption of a common developmental regulator such as NR5A1 was hypothesized. The patient was found to have a de novo heterozygous p.G35E change in the P-bo of NR5A1, which is important in dictating DNA binding specificity through its interaction with DNA response elements in the regulatory regions of target genes (92).

The second report of NR5A1 defects in humans was described by Biason-Lauber and Schoenle, in a 14-month-old 46,XX girl who had presented with primary adrenal insufficiency and seizures (93) . She had a de novo heterozygous NR5A1 change resulting in the p.R255L variant into the proximal part of the ligand-like binding domain of the protein. The ovaries were detected by MRI scan and Inhibin A levels were normal for her age, suggesting that NR5A1 change had not disrupted ovarian function. The follow up of this girl until 16.5 years old showed a normal puberty and regular menstruation showing that phenotypic variant of NR5A1 allelic variant in a 46, XX affected person includes adrenocortical insufficiency but no ovarian dysfunction at pubertal age (94).

The third report of NR5A1 defects in humans was found in an infant with a similar phenotype of the first case: primary adrenal failure and 46,XY DSD. However, this child had inherited the homozygous p.R92Q alteration in a recessive manner (95). The change lies within the A-box of NR5A1, which interferes with monomeric DNA binding stability, but in vitro functional activity was in the order of 30–40% of the wild type (95-97). Carrier parents showed normal adrenal function suggesting that the loss of both alleles is required for the phenotype development when disrupted protein keeps this level of functional activity. In addition, another family has been reported with a homozygous missense variant (p.D293N) in the LBD of NR5A1 (98). This change also showed partial loss-of-function (50%) in gene transcription assays.

In 2004, we reported the fourth NR5A1 deleterious variant in humans, which brought two novel variables to the NR5A1 phenotype: it was the first frameshift variant and it was identified in a 34-year-old 46,XY DSD female with normal adrenal function (99). Another interesting aspect in this patient was the absence of gonadal tissue at laparoscopy. Since she had atypical genitalia and absence of Müllerian derivatives, we assumed that testicular tissue regressed completely late in fetal life.

NR5A1 changes associated with 46,XY DSD are usually frameshift, nonsense or missense changes that affect DNA-binding and gene transcription (96). Most of the point variants identified in NR5A1 are located in the DNA-binding domain of the protein. The p.L437Q variant, the first located in the ligand-binding region, was identified in a patient with a mild phenotype, a penoscrotal hypospadias. This protein retained partial function in several NR5A1-expressing cell lines and its location points to the existence of a ligand for NR5A1, considered an orphan receptor so far (97). NR5A1 is bound to sphingosine (SPH) and lyso-sphingomyelin (lysoSM) under basal conditions (100,101).

Progressive androgen production and virilization in adolescence has been observed in several XY patients with NR5A1 variants, in contrast to the severe under virilized external genitalia found in most patients (101,102). The almost normal testosterone levels after hCG stimulation test or at pubertal age suggest that NR5A1 action might be less implicated in pubertal steroidogenesis than during fetal life.

In contrast, fetal Sertoli cell function seems to be preserved in most patients with heterozygous NR5A1 variants based on the common observation of absent Müllerian derivatives and primitive seminiferous tubules in histology. The reviewed data of seventy-two 46,XY DSD patients with NR5A1 pathogenic variants reported in the literature, for whom information on the presence or absence of Müllerian derivatives was available, suggested that Müllerian derivatives are present in about 24% of the cases. However, persistently elevated FSH levels after puberty found in all patients studied suggest an impairment of Sertoli cells function in post pubertal age.

More than 180 different NR5A1 variants, distributed across the full length of the protein, have been described and the majority are nonsynonymous variants (103-105).

Most of these variants are in the DNA binding domain and are in heterozygous state or compound heterozygous state with the p.G46A (rs1110061) variant. A clear correlation between the location of a variant, it’s in vitro functional performance and the associated phenotype is not observed. Indeed, family members bearing the same NR5A1variant may present with different phenotypes (106).

The contribution of other genetic modifiers has been suggested to explain phenotypic variability. Exome sequencing analyses of DSD patients have identified pathogenic variants or variants of uncertain significance in several genes involved in sexual development (42). In a 46,XY patient with atypical external genitalia, palpable inguinal gonads, absent uterus in pelvic ultrasonography and poor testosterone response to hCG stimulation, Mazen and colleagues identified, by exome sequencing, the previously described p.Arg313Cys NR5A1 variant in compound heterozygous state with a p.Gln237Arg MAP3K1 variant 27169744 (107). This NR5A1 variant was previously reported in association with mild hypospadias (108), and a possible digenic inheritance was proposed to explain the phenotypic heterogeneity (107).

In several cohort studies, NR5A1 changes have been reported in approximately 10–15% of the individuals with gonadal dysgenesis (89,96). Although many of the heterozygous changes are de novo, about one-third of these changes have been shown to be inherited from the mother in a sex-limited dominant manner (96). These women are at potential risk of primary ovarian insufficiency but while fertile they can pass NR5A1 heterozygous changes to their children. This mode of transmission can mimic X-linked inheritance (96). The features in different affected family members can be variable.

A different role of NR5A1 in human reproductive function was described by Bashamboo and co-workers (109). They investigated whether changes in NR5A1 could be found in a cohort of 315 men with normal external genitalia and non-obstructive male factor infertility where the underlying cause was unknown (109). Analysis of NR5A1 in this cohort identified heterozygous changes in seven individuals; all of them were located within the hinge region of the NR5A1 protein. The men who harbored NR5A1 changes had more severe forms of infertility (azoospermia, severe oligozoospermia) and in several cases low testosterone and elevated gonadotropins were found. A serial decrease in sperm count was found in one-studied men, raising the possibility that heterozygous changes in NR5A1 might be transmitted to offspring, especially if fatherhood occurs in young adulthood rather than later in life (110). As progressive gonadal dysgenesis is likely, gonadal function should be monitored in adolescence and adulthood, and early sperm cryopreservation considered in male patients, if possible. In conclusion, this study shows that changes in NR5A1 may be found in a small subset of phenotypically normal men with non- obstructive male factor infertility where the cause is currently unknown. These individuals may be at risk of low testosterone in adult life and may represent part of the adult testicular dysgenesis syndrome (110,111).

A novel heterozygous missense variant (p.V355M) in NR5A1 was identified in one boy with a micropenis and testicular regression syndrome (112). NR5A1 variants have also been identified in familial and sporadic forms of 46,XX primary ovarian insufficiency not associated with adrenal failure (98,113). Most of these women harbored heterozygous alterations in NR5A1 and had been identified in families with histories of 46,XY DSD and 46,XX POI. Heterozygous NR5A1 changes were also found in two girls with sporadic forms of POI (98). In one large kindred, a partial loss-of-function NR5A1 change (p.D293N) was inherited in an autosomal recessive manner. These 46,XX women with p.D293N NR5A1 variant presented with either primary or secondary amenorrhea and with a variable age of features onset. The detection of NR5A1 alterations in 46,XX ovarian failure shows that NR5A1 is also a key factor in ovarian development and function in humans. Thus, some 46,XX women with NR5A1 variants have normal ovarian function and can transmit the variant in a sex-limited dominant pattern. Therefore, the inheritance patterns associated with NR5A1 changes can be autosomal dominant, autosomal recessive or sex-limited dominant.

NR5A1 defects can be found in association with a wide range of human reproductive phenotypes such as 46,XY and 46,XX disorders of sex development (DSD) associated or not with primary adrenal insufficiency, male infertility, primary ovarian insufficiency and finally testicular or ovo-testicular 46,XX DSD (101) (103) (Table 4). Spleen development anomalies have been described in patients with NR5A1 variants (103).

*Table 2. Spectrum of Phenotypes Caused by NR5A1* Defects**
Karyotype	Phenotypes	Number of reported patients	Reference
46,XY	DSD and adrenal insufficiency	2	(92,95)
	DSD without adrenal insufficiency	69	(63,89,96,98,100,101,103,106)
	Male infertility	10	(63)
	Ovotesticular DSD and genitopatellar syndrome*	1	(114)
46,XX	Adrenal insufficiency	2	(92,93)
	Female infertility, POI	14	(98,101,103)
	(Ovo) testicular DSD without adrenal insufficiency	11	(98,115)

**46,XY DSD Due to Under Expression of the SRY Gene**

Most of the authors reported pathogenic allelic variants in SRY gene in less than 20% of the patients with complete 46,XY gonadal dysgenesis (116-118). In the partial form, the frequency of SRY variants is even lower than in the complete form. To date, most of the SRY variants are located in the HMG box, showing the critical role of this domain, and are predominantly de novo variants. However, some cases of fertile fathers and their XY affected children, sharing the same altered SRY sequence, have been reported (116,119). In a few of these cases, the father’s somatic mosaicism for the normal and mutant SRY gene has been proven (120) The variable penetrance of SRY variants in familial cases have been described in SRY mutant proteins with relatively well preserved in vitro activity (121).

**Dysgenetic 46,XY DSD Associated with Campomelic Dysplasia (Under Expression of the SOX9 Gene)**

SRY-related HMG-box gene 9 (SOX9) is a transcription factor involved in chondrogenesis and sex determination. SOX9 gene, located on human chromosome 17, is a highly conserved HMG family member and it is also implicated in the male sex-determining pathway (122,123).

Pathogenic allelic variants in SOX9 have been identified in heterozygous state in patients with Campomelic dysplasia (122). This syndrome is characterized by severe skeletal malformations associated with dysgenetic 46,XY DSD. These patients have variable external genitalia ranging from that of normal male with cryptorchidism to atypical or female genitalia, and the internal genitalia may include vagina, uterus, and fallopian tubes (124).

Intact SOX9 were also reported in patients with Campomelic dysplasia and 46,XY gonadal dysgenesis. The genomic analysis of the SOX9 locus in these patients identified a key regulatory element termed RevSex, located approximately 600 kb upstream from SOX9. RevSex is duplicated in individuals with 46,XX (ovo)testicular DSD and deleted in individuals with 46,XY GD (125,126). Moreover, structural changes involving multiple regions both upstream and downstream of the SOX9 gene have been associated with non-syndromic XY DSD (127,128). These findings indicate that variants located in the regulatory elements of SOX9 should be routinely screened in a DSD diagnostic setting (69).

Dysgenetic 46,XY DSD Due to Under Expression of the FGF9/FGFR2 Genes

The importance of Fgf9/Fgfr2 signaling pathway in mouse testis determination is well known (129,130). In the developing testis occurs a positive feedback loop among Fgf9/Fgfr2/Sox9; Fgf9 is upregulated by Sox9 and signals through Fgfr2 maintain Sox9 expression (129) and this loop represses Wnt4 (131).

Mice homozygous for a null variant in Fgf9 or Fgfr2 exhibit male-to-female sex reversal, with all testis-specific cellular events being disrupted, including cell proliferation, mesonephric cell migration, Sertoli cell differentiation, and testis cord formation (129,130,132). However, in human sex development the role of FGF9 and FGFR2 remains unclear.

In humans, the only reported pathogenic variants in FGF9 are associated with craniosynostosis or multiple synostosis phenotypes, and no FGF9 variants were identified in 46,XY GD patients (133).

Human FGFR2 variants have been related with some syndromes as lacrimo-auriculo-dento-digital, characterized by tear tract, ear, teeth and digit abnormalities (133) and craniosynostosis syndromes including Crouzon, Pfeiffer, Apert and Antley-Bixler syndromes (134-136). FGFR2 variants can lead to loss (LAAD syndrome) or gain (craniosynostosis syndromes) of function in these disorders (137). No gonadal defects were described in patients with LADD or craniosynostosis syndromes.

A single 46,XY patient with gonadal dysgenesis and craniosynostosis was described by Bagheri-Fam et al (138). This patient had abnormalities which are identified in several craniosynostosis syndromes (short stature, brachycephaly, proptosis, down slanting palpebral fissures, low-set dorsally rotated ears, reduced extension at the elbows but absence of hand and feet anomalies). She also presented female external genitalia, primary amenorrhea and gonadal dysgenesis with dysgerminoma. DNA sequencing revealed a cysteine-to-serine substitution at position 342 in the FGFR2c isoform (p.C342S). Cys342 substitutions by Ser or other amino acids (Arg/Phe/Trp/Tyr) occur frequently in the craniosynostosis syndromes Crouzon and Pfeiffer but these patients do not present gonadal abnormalities. Variants in the 2c isoform of FGFR2 is in agreement with knockout data showing that FGFR2c is the critical isoform during sex determination in the mouse. Taken together, these data suggest that the FGFR2c c.1025G>C (p.C342S) variant might contribute to 46,XY DSD in this patient. The authors proposed that this heterozygous variant leads to gain of function in the skull, but to loss of function in the developing gonads and that she might harbor a unique set of modifier genes, which exacerbate this testicular phenotype (138).

The authors proposed that the p.C342S heterozygous variant in FGFR2c leads to gain of function in the skull, but loss of function in the developing gonads; and that the presence of modifier genes would exacerbate the testicular phenotype in this patient (138). However, the presence of a pathogenic variant involving other DSD genes, cannot be completely excluded in this patient.

Dysgenetic 46,XY DSD Due to Disruption in Hedgehog Signaling

DESERT HEDGEHOG (DHH) GENE

It is a member of the hedgehog family of signaling proteins, is located in chromosome 12-q13.1 and is one of the genes involved in the testis-determining pathway (139). Dhh seems to be necessary for Nr5a1 up-regulation in Leydig cells in mice (140). To date, six homozygous variants have been described in DHH gene in 46,XY patients conferring phenotypes ranging from partial to complete gonadal dysgenesis, associated or not with polyneuropathy. The first one, the homozygous missense variant (p.M1T) is located at the initiation codon of exon 1 and was found in a 46,XY patient with partial gonadal dysgenesis associated with polyneuropathy (141). Two other variants, one the p.L162P located at exon 2 and the other the p.L363CfsX4 located in exon 3 were identified in three patients with complete gonadal dysgenesis without polyneuropathy; two of them harbored gonadal tumors (bilateral gonadoblastoma and dysgerminoma, respectively) (142). Later, the c.1086delG variant was identified in heterozygous state in two patients with partial gonadal dysgenesis (143). In addition, two novel homozygous variants were described in two patients with complete 46,XY gonadal dysgenesis without clinically overt polyneuropathy (144). In both sisters, clinical neurological examination revealed signs of a glove and stocking like polyneuropathy. The first defect, the c.271_273delGAC resulted in deletion of one amino acid (p.D90del) and the second one, a duplication c.57_60dupAGCC resulted in a premature termination of DHH protein (144) . The p.R124Q variant was identified by exome sequencing in two sisters of a consanguineous family with 46, XY gonadal dysgenesis and testicular seminoma (145).

HEDGEHOG ACETYL-TRANSFERASE (HHAT) GENE

The HHAT protein is a member of the MBOAT family of membrane-bound acyl-transferases which catalyzes amino-terminal palmitoylation of Hh proteins. The novel variant (p.G287V) in the HHAT gene was found in a syndromic 46,XY DSD patient with complete gonadal dysgenesis and skeletal malformation by exome sequencing. This variant disrupted the ability of HHAT protein to palmitoylated Hh proteins including DHH and SHH (146) In mice, the absence of Hhat in the XY gonad did not affect testis-determination, but impaired fetal Leydig cells and testis cords development (146). The phenotype of the girl carrying the homozygous p.G287V variant is a rare combination of gonadal dysgenesis and chondrodysplasia. Moreover, a de novo dominant variant in the MBOAT domain of HHAT was reported in association with intellectual disability and apparently normal testis development (147).

46,XY DSD Due to Under Expression of the DMRT1 Gene

Raymond et al identified both DNA-binding Motif (DM) domain genes expressed in testis (DMRT1 and DMRT2) located in chromosome 9p24.3, a region associated with gonadal dysgenesis and 46,XY DSD (148-150). The human 9p monosomy syndrome is characterized by variable degrees of 46,XY DSD, from female genitalia to male external genitalia with cryptorchidism associated to agonadism, streak gonads or hypoplastic testes and internal genitalia disclosing normal Müllerian or Wolffian ducts, mental retardation and craniofacial abnormalities (151). Gonadal function varies from insufficient to near normal testicular production. It is inferred that haploinsufficiency of DMRT1and DMRT2 primarily impairs the formation of the undifferentiated gonad, leading to various degrees of testis or ovary formation defects (151).

Although 9p24 deletions are a relatively common cause of syndromic 46,XY gonadal dysgenesis, the pathogenic variants within DMRT1 are rarely identified (152).

Genomic–wide copy number variation screening revealed that DMRT1 deletions were associated with isolated 46,XY gonadal dysgenesis in addition to inactivation variants (133,148). In vitro studies to analyze the functional activity of the DMRT1 (p.R111G) variant identified by exome sequencing in a patient with 46,XY complete gonadal dysgenesis, indicated that this protein had reduced DNA affinity and altered sequence specificity. This mutant DMRT1, when mixed with the wild-type protein bound as a tetramer complex to an in vitro Sox9 DMRT1-binding site, differs from the wild-type DMRT1 that is usually bound as a trimer. This suggests that a combination of haploinsufficiency and a dominant disruption of the normal DMRT1 target binding site is the cause of the abnormal process of testis-determination seen in this patient (153).

Matson et al. (2011) have shown in mice that Dmrt1 and Foxl2 create another regulatory network necessary for maintenance of the testis during adulthood. Loss of Dmrt1 in mouse Sertoli cells induces the reprogramming of those into granulosa cells, due to Foxl2 upregulation. Consequently, theca cells are formed, estrogens are produced, and germ cells appear feminized (49).

ATR-X Syndrome (X-linked α-Thalassemia and Mental Retardation)

ATR-X syndrome results from variants in the gene that encodes for X-linked helicase-2, implicating ATR-X in the development of the human testis (154). Genital anomalies leading to a female sex of rearing were reported in several affected 46,XY patients with ATR-X syndrome (155).

ATR-X syndrome is characterized by severe mental retardation, alpha thalassemia and a range of genital abnormalities in 80% of cases (154). In addition to these definitive phenotypes, patients also present with typical facial anomalies comprising a carp-like mouth and a small triangular nose, skeletal deformities and a range of lung, kidney, and digestive problems. A variety of phenotypically overlapping conditions (Carpenter-Waziri syndrome, Holmes-Gang syndrome, Jubert-Marsidi syndrome, Smith-Fineman-Myers syndrome, Chudley-Lowry syndrome and X-linked mental retardation with spastic paraplegia without thalassemia) have also been associated with ATRX variants (154).

ATRX lies on the X chromosome (Xq13) and the disease has been confined to males; in female carriers of an ATRX variant, the X-inactivating pattern is skewed against the X chromosome carrying the mutant allele.

Urogenital abnormalities associated with variants in human ATRX range from undescended testes to testicular dysgenesis with female or atypical genitalia. Duplication of Xq12.2-Xq21.31 that encompasses ATRX along with other genes has been described in a male patient with bilateral cryptorchidism and severe mental retardation. The patient entered spontaneous puberty by the age of 12 and developed bilateral gynecomastia (156). There are two major functional domains in ATRX protein: 1- the ATRX-DNMT3-DNMT3L (ADD) domain at the N-terminus and 2- the helicase/ATPase domain at the C-terminal half of the protein, both acting as chromatin remodeling. variants in the ADD domain have been related to severe psychomotor impairment associated with urogenital abnormalities. On the other hand, variants in the C-terminus region have been related with mild psychomotor impairment without severe urogenital abnormalities (157,158).

Although all cases of severe genital abnormality reported in ATRX syndrome have been associated with severe mental retardation, this is not true for alpha-thalassemia. The role of ATRX in the sexual development cascade is poorly understood and it is suggested that it could be involved in the development of the Leydig cells (159).

**Dysgenetic 46,XY DSD Due to Under Expression of the MAP3K1 Gene**

MAPK signaling pathway role in mammalian sex-determination is still poorly understood. In mice, it has been shown that the Map3k4 gene is essential for testicular determination, since the lack of activity of this protein leads to failure of testicular cord development and disorganization of gonadal tissue in development (160). In mice, the reduction of the Gadd45/Map3k4/p38 pathway activity is associated with a reduction in the Sry expression in the XY mice gonad at sex-determination causing sex-reversal in these animals (161). Studies with knock-in animals for the Map3k1 gene demonstrated a lower repercussion in the testicular tissue, which present a reduction in the Leydig cells number (162,163). However, in patients with 46, XY gonadal dysgenesis, different non-synonymous allelic variants were identified in the MAP3K1 gene. The first variant described was identified for mapping by linkage analysis of an autosomal sex-determining gene locus at the long arm of chromosome 5 in two families with 46,XY DSD, including patients with complete and partial gonadal dysgenesis. The splice-acceptor variant c.634-8T>A in the MAP3K1 disrupted RNA splicing and was segregated with the phenotype in the first family. Variants in the MAP3K1 were also demonstrated in the second family (p.G616R) and in two of 11 sporadic 46,XY DSD patients (p.L189P, p.L189R) studied (51,164). Subsequently, the two novel variants p.P153L and c.2180- 2A>G in the MAP3K1 were identified in non-syndromic patients with 46,XY gonadal dysgenesis. Functional studies of mutated MAP3K1 proteins identified change in phosphorylation targets in subsequent steps of the cascade of MAP3K1, p38 and ERK1/2 and enhanced the binding of the Ras homolog gene family, member A (RHOA) to the MAP3K1 complex (51). In normal male gonadal development, the binding of MAP3K1 to the RHOA protein promotes a normal phosphorylation of p38 and ERK1/2, and a blockade of the β-catenin pathway is determined by MAP3K4. In the female development, hyperphosphorylation of p38 and ERK1/2 occurs and the presence of p38 and ERK1/2 hyperphosphorylated determine the activation of the β-catenin pathway, that result in a block of the positive feedback pathway of SOX9 and the testicular development (51) .

Cohorts of patients with 46,XY DSD studied by a targeted gene panel have found several new potentially deleterious variants and uncertain significance variants in the MAP3K1 (26). Although the findings strongly indicate the participation of the MAP3K1 variants in the etiology of testicular development abnormalities, a better understanding of the mechanisms of MAPK pathway in the gene regulatory networks of the human testicular determination process is still necessary (52,107).

**46,XY DSD Due to Over Expression of the NR0B1/DAX1 Gene**

Male patients with female or atypical external and internal genitalia due to partial duplications of Xp in the presence of an intact SRY gene have been described (28). These patients present with dysgenetic or absent gonads associated or not with mental retardation, cleft palate, and dysmorphic face. Bardoni et al. identified in these patients, a common 160-kb region of Xp2 containing NR0B1/DAX1 gene named dosage sensitive sex locus which, when duplicated, resulted in 46,XY DSD (164).

The large duplications of Xp21 reported prior to array-CGH and MLPA techniques were identified by conventional karyotyping. Patients carried large genomic rearrangements involving several genes. In these patients, the presence of XY gonadal dysgenesis was part of a more complex phenotype, which also included dysmorphic features and/or mental retardation (165).

Interestingly, in all cases with isolated 46,XY gonadal dysgenesis, the IL1RAPL1 gene located immediately to the duplication containing NR0B1/DAX1, is not disrupted. Deletions or variants of this gene have been identified in patients with mental retardation (166). Disruption of this gene could explain the mental retardation previously described in patients with larger Xp21 duplications (167).

Several patients with isolated 46,XY gonadal dysgenesis due to duplications of Xp21 have been described. The first report identified a 637 kb tandem duplication on Xp21.2 that in addition to NR0B1/DAX1 includes the four MAGEBgenes in two sisters with isolated 46,XY gonadal dysgenesis and gonadoblastoma (168). The second case exhibited a duplication with approximately 800 kb in size and, in addition to NR0B1/DAX1, contains the four MAGEB, Cxorf21 and GK genes. The healthy mother was a carrier of the duplication (169).

Smyk et al. described a 21-years-old 46,XY patient manifesting primary amenorrhea, a small immature uterus, gonadal dysgenesis and absence of adrenal insufficiency with a submicroscopic deletion (257 kb) upstream of NR0B1/DAX1. The authors hypothesized that loss of regulatory sequences may have resulted in up-regulation of DAX1 expression, consistent with phenotypic consequences of NR0B1/DAX1 duplication (170).

By using array-CGH and MLPA techniques, additional NR0B1/DAX1 locus duplications have been identified in patients with isolated 46,XY gonadal dysgenesis (28,169,171).

Barbaro et al. identified a relatively small NR0B1/DAX1 locus duplication responsible for isolated complete 46,XY gonadal dysgenesis in a large English family (28). The duplication extends from the MAGEB genes to part of the MAP3K7IP3 gene, including NR0B1, CXorf21, and GK genes. Unfortunately, the authors were unable to set up the rearrangement mechanism and distinguish between a nonallelic homologous recombination or a nonhomologous end joining mechanism. Therefore, until now, there is not a direct proof that an isolated NR0B1/DAX1 duplication is sufficient to cause 46,XY gonadal dysgenesis in humans, suggesting that other contiguous genes located in the DSS locus, should be involved in dosage-sensitive 46,XY DSD.

X-inactivation patterns in fertile female carriers of each of the three small NR0B1 locus duplications were analyzed (169). They established that female carrier of macroscopic Xp21 duplications are healthy and fertile due to the preferential inactivating of the duplicated chromosome and thereby protecting them from increased NR0B1 expression (169).

**46,XY DSD Due to the Over Expression of WNT4 Gene**

The WNT4 (wingless-type mouse mammary tumor virus integration site member 4) gene belongs to a family that consists of structurally related genes that encode cysteine-rich secreted glycoproteins that act as extracellular signaling factors (172).

Overexpression of the WNT4 and RSPO1 may be a cause of 46,XY DSD. A 46,XY newborn infant, with multiple congenital anomalies including bilateral cleft lips and palate, intrauterine growth retardation, microcephaly, tetralogy of Fallot, atypical external and internal genitalia, and undescended gonads consisted of rete testes and rudimentary seminiferous tubules, who carried a duplication of 1p31-p35, including both WNT4 and RSPO1 genes, was reported (173). In vitro functional studies showed that Wnt4 up-regulates Nr0b1/Dax1 in Sertoli cells, suggesting that Nr0b1/Dax1 overexpression was the cause of 46,XY DSD in this infant (174).

Table 3. Phenotypic Spectrum of Defects in the Genes Involved in Human Male Sex Determination
Genes	Chromosome position	Molecular defect	External genitalia	Müllerian ducts derivatives	Testes	Associated anomalies	Associated Syndrome
ARX	Xp22	Deletion/ Inactivating variants	Atypical/ micropenis with cryptorchidism	-	Dysgenetic	Abnormal psychomotor development, epilepsy, spasticity, and intellectual disability	X-linked lissencephaly, Proud syndrome, Ohtahara syndrome
ATRX	Xq13	Inactivating variants	Atypical / Male with cryptorchidism	-	Dysgenetic	Severe psycho-motor retardation, dysmorphic face, cardiac and skeletal abnormalities, thalassemia	Alpha thalassemia and mental retardation X-linked
CBX2	17q25	Inactivating variants	Female	+	Normal Ovary	No	No
DHH	12q12	Inactivating variants	Female/Atypical	+/-	Dysgenetic / Testis	Minifascicular neuropathy	No
DHX37	12q24.31	Inactivating variants	Male with cryptorchidism and micropenis, Atypical	+/-	Dysgenetic/ Absent	No	No
DMRT1	9p24	Deletion/Inactivating variants	Female/ Atypical/ Male with cryptorchidism	+/-	Dysgenetic/Absent/ Hypoplastic	Craniofacial Abnormalities, microcephaly, mental retardation	No
DSS locus (DAX-1 /MAGEB)	Xp21	Gene duplication	Female/ Atypical/ male	+/-	Dysgenetic/ Absent	Mental retardation, cleft palate, dysmorphic face	No
FGFR2	10q26	Inactivating variants	Female	ND	Dysgenetic	Short stature, craniofacial abnormalities, elbow and knee contractures	Craniosynostosis syndrome
FOG2/ZFPM2	8q23	Balanced translocation, inactivating variants	Male	-	Probable dysgenetic	Heart defects	No
GATA4	8p23	Inactivating variants	Atypical / male with micropenis	-	Normal/ Dysgenetic	Heart defects	No
HHAT	1q32	Inactivating variants	Female	+	Dysgenetic	Chondrodysplasia	Nivelon-Nivelon-Mabille syndrome
MAP3K1	5q11.2	Inactivating mutation	Female/Atypical	+	Dysgenetic	No	No
MYRF	11q12.2	Inactivating variants	Female/Atypical	-	ND	Congenital heart defects, urogenital anomalies, congenital diaphragmatic hernia, and pulmonary hypoplasia	Cardiac urogenital syndrome
NR5A1	9q33	Inactivating variants	Female/Atypical/ Male with cryptorchidism Male with spermatogenic failure	+/-	Normal/ Dysgenetic/ Absent	Adrenal Insufficiency	No
PPP1R12A	12q21.2- q21.31	Inactivating variants	Female/Atypical	+/-	Dysgenetic	Genitourinary and/or brain malformations	No
SOX9	17q24.3-25.1	Inactivating variants, 5’ and 3’ Rearrangements	Female/ Atypical Male	+/-	Dysgenetic	Severe skeletal defects	Campomelic displasia
SRY	Yp11.3	Inactivating variants	Female/ Atypical	+	Dysgenetic	No	No
WNT4 /RSPO1 locus	1p34.3-p35	Gene duplication	Atypical	+	Dysgenetic	Cleft lips and palate, tetralogy of Fallot, intrauterine growth retardation, microcephaly	No
WT1	11p13	Inactivating variants	Female/ Atypical	+/-	Dysgenetic	Late-onset renal failure Gonadoblastoma	Frasier
		Inactivating variants	Atypical	+/-	Dysgenetic	Early-onset renal failure, Wilm's tumor	Denys-Drash
		Inactivating variants	Female/ Atypical / Male with cryptorchidism	-	Dysgenetic	Mental retardation, Wilm's tumor, Aniridia, renal agenesis or horseshoe kidney	WAGR
WWOX	16q23	Multi-exons deletion	Atypical	+	Dysgenetic	No	-

ND: data not described

46,XY DSD ASSOCIATED WITH CHOLESTEROL SYNTHESIS DEFECTS

Smith-Lemli-Opitz Syndrome (SLOS)

This syndrome, caused by a deficiency of 7-dehydrocholesterol reductase, is the first true metabolic syndrome leading to multiple congenital malformations (179,180).

This disorder is caused by variants in the sterol delta-7-reductase (DHCR7) gene, which maps to 11q12-q13. Typical facial appearance is characterized by short nose with anteverted nostrils, blepharoptosis, microcephaly, photosensitivity, mental retardation, syndactyly of toes 2 and 3, hypotonia, and atypical genitalia. Adrenal insufficiency may be present or evolve with time. Atypical external genitalia are a frequent feature of males (71%) and ranges from hypospadias to female external genitalia despite normal 46,XY karyotype and SRY sequences. Müllerian derivative ducts can also be present (181-183). The etiology of masculinization failure in SLOS remains unclear. However, the description of patients with SLOS who present with hyponatremia, hyperkalemia, and decreased aldosterone-to-renin ratio suggest that the lack of substrate to produce adrenal and testicular steroids is the cause of adrenal insufficiency and atypical genitalia (184), although, a revision of HPA axis in these patients showed normal HPA axis function (185).

Affected children present elevations of 7-dehydrocholesterol (7DHC) in plasma or tissues. 7DHC is best assayed using Gas Chromatography/Mass Spectroscopy (GC/MS). Considering the relative high frequency of Smith-Lemli-Opitz syndrome, approximately 1 in 20,000 to 60,000 births, we suggest that at least cholesterol levels should be routinely measured in patients with 46,XY DSD. However, although frequently low, plasma cholesterol levels can be within normal limits in affected patients.

DHCR7 variant analysis can confirm a diagnosis of SLOS. The human DHCR7 gene is localized on chromosome 11q13 and contains nine exons encoding a 425 amino-acid protein (64). More than 130 different variants of DHCR7have been identified and the great majority of them are located at the exons 6 to 9 (186,187). However, the genotype-phenotype correlation in SLOS is relatively poor (188).

Currently, most SLOS patients are treated with cholesterol supplementation that can be achieved by including high cholesterol foods and/or suspensions of pharmaceutical grade cholesterol. Data suggests that early intervention may be of benefit to SLOS patients (189). Observational studies report improved growth and muscle tone and strength, increased socialization, decreased irritability and aggression in SLOS patients treated with cholesterol supplementation. However, in a group of SLOS patients’ treatment with a high cholesterol diet did not improve developmental scores (190).

Treatment with simvastatin, an HMG-CoA reductase inhibitor, aiming to block the cholesterol synthesis pathway avoiding the formation of large amounts of 7DHC/8DHC, and in this manner limiting exposure to potentially toxic metabolites in SLOS patients has been proposed. Simvastatin can also cross the blood–brain barrier and may provide a means to treat the biochemical defect present in the CNS of SLOS patients (191). A major effect of statin therapy is the transcriptional upregulation of genes controlled by the transcriptional factor SREBP, including DHCR7. Thus, if any residual activity is present in the mutant DHCR7, its upregulation could increase intracellular cholesterol synthesis. Simvastatin use in SLOS patients resulted in a paradoxical increase in serum and cerebrospinal fluid cholesterol levels (191). Randomized controlled-placebo trials were performed with simvastatin in SLOS showing significant reduction in plasmatic 7DHC associated with improvement in irritability symptoms (192). Determination of residual DHCR7 enzymatic activity may be helpful in selecting SLOS patients to be considered for a beneficial response of statins (187). Recently, promising gene therapy using an adeno-associated virus vector carrying a functional copy of the DHCR7 gene was administered by intrathecal injection in a mouse model with improvement of cholesterol levels in the central nervous system (193).

Table 4. Phenotype of 46,XY Subjects with Smith-Lemli-Optiz Sndrome
Inheritance	Autosomal recessive
External genitalia	Micropenis and/or hypospadias, hypoplastic or bifid scrotum; female
Müllerian duct derivatives	May be present
Wolffian duct derivatives	Absent to male
Testes	Scrotum, inguinal or intra-abdominal region
Clinical features	Facial and bone abnormalities. Heart and pulmonary defects. Renal agenesis. Mental retardation, Seizures, hypotonia, syndactyly of second and third toes.
Puberty	Apparently normal
Hormonal diagnosis	Low cholesterol, elevated 7-dehydrocholesterol. Decreased aldosterone-to-renin ratio
Gender role	Male
DHCR7 gene location	11q12-q13
Molecular defect	variants in DHCR7 gene
Treatment	Dietary cholesterol supplies accompanied by ursodeoxycholic acid, and statins
Outcome	Severe mental retardation

Dysgenetic 46,XY DSD Due to Under Expression of the DHX37 gene

46,XY gonadal dysgenesis (GD) is a heterogeneous group of disorders with a wide phenotypic spectrum, including embryonic testicular regression syndrome (ETRS) (Table 5). Screening of 87 patients with 46,XY DSD (17 familial cases from 8 unrelated families and 70 sporadic cases) using whole-exome sequencing and target gene-panel sequencing identified a new player in the complex cascade of male gonadal differentiation and maintenance - the Asp-Glu-Ala-His-box (DHX) helicase 37 (DHX37) gene (53). The variants were especially associated with ETRS (7/14 index cases; 50%). The frequency of rare, predicted-to-be-deleterious DHX37 variants in this cohort (14%) is significantly higher than that observed in the Genome Aggregation Database (0.4%; P < 0.001). Immunohistochemistry analysis in human testis showed that DHX37 is mainly expressed in germ cells at different stages of testis maturation, in Leydig cells, and rarely in Sertoli cells. Other papers confirmed these findings, associating 46,XY gonadal dysgenesis with defects in DHX37 gene (152,175).

*Table 5. Phenotype of 46,XY Subjects with Gonadal Dysgenesis Due to DHX37Defects*
Inheritance	Autosomal dominant
External genitalia	Micropenis, atypical genitalia or typical female
Müllerian duct derivatives	Absent uterus, Fallopian tubes may be present
Wolffian duct derivatives	Present
Testes	Abdominal region or absent
Histological analysis	Dysgenetic, no gonadal tissue
Puberty	Hypergonadotropic hypogonadism
Hormonal diagnosis	Elevated serum levels of LH and FSH; very low levels of testosterone and normal testosterone precursors levels
Gender role	Male, female, male to female
DHX37 gene location	12q24.31
Molecular defect	Heterozygous variants in DHX37 gene
Treatment	Repair of atypical genitalia; estrogen or testosterone replacement according to social sex
Outcome	Most patients keep the male social sex; some change to female social sex

Different modes of inheritance have been reported in familial cases of 46,XY gonadal dysgenesis, including autosomal dominant, autosomal recessive, X-linked and multifactorial inheritance (polygenic) (107,176-178). Oligogenic mode of inheritance might explain genotype/phenotype variability observed in 46,XY gonadal formation patients. Pathogenic allelic variants in NR5A1, DHX37, MAP3K1 and SRY are the most frequent molecular causes of 46,XY gonadal dysgenesis (20).

46,XY DSD DUE TO TESTOSTERONE PRODUCTION DEFECTS

46,XY DSD Due to Impaired Leydig Cell Differentiation (Complete and Partial Forms)

Inactivating variants of human LHCG receptor (LHCGR) have been described in 46,XY individuals with a rare form of disorder of sex development, termed Leydig cell hypoplasia. These inactivating variants in the LHCGR prevent LH and hCG signal transduction and thus testosterone production both pre- and postnatally in genetic males (194).

Both hCG and LH act by stimulating a common transmembrane receptor, the LHCGR (195) LHCGR is a member of G protein-coupled receptors, which are characterized by the canonical serpentine region, composed of seven transmembrane helices interconnected by three extracellular and three intracellular loops (196,197). The large amino-terminal extracellular domain, rich in leucine-repeats, mediates the high affinity binding of pituitary LH or placental human chorionic gonadotropin (hCG) (197).

LHCGR activates the Gs protein, which determines an increase in intracellular cAMP and a subsequent stimulation of steroidogenesis in gonadal cells such as testicular Leydig cells, ovarian theca cells and differentiated granulosa cells (195,198) A secondary mechanism of LHCGR stimulation is through G_q/11 protein activation and the inositol phosphate signaling pathway (197).

The LHCGR gene is located on the short arm of chromosome 2 (2p21). It spans nearly 80 kb and has been thought to be composed of 11 exons and 10 introns. Exon 11 of the LHCGR gene encodes the entire serpentine domain as well as the carboxy-terminal portion of the hinge region (NCBI GeneID 3973; http://www.ncbi.nlm.nih.gov). The amino-terminal portion of the hinge region is encoded by exon 10 and the signal peptide and remaining portion of the extracellular domain are encoded by exons 1-9 (194,196). A novel primate-specific exon (termed exon 6A) was identified within intron 6 of the LHCGR gene. This exon is not used by the wild-type full-length receptor. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay in LHCGR. When exon 6A is utilized, it results in a truncated LHCGR protein (199).

In 1976, Berthezene et al. (200) described the first patient with Leydig cell hypoplasia and subsequently several cases have been reported (201-203). The clinical features are heterogeneous and result from a failure of intrauterine and pubertal virilization. A review of the literature allowed delineation of the characteristics of 46,XY DSD due to the complete form of Leydig cell hypoplasia as: 1) female external genitalia leading to female sex assignment 2) no development of sexual characteristics at puberty, 3) undescended testes slightly smaller than normal with relatively preserved seminiferous tubules and absence of mature Leydig cells, 4) presence of rudimentary epididymis and vas deferens and absence of uterus and fallopian tubes, 5) low testosterone levels despite elevated gonadotropin levels, with elevated LH levels predominant over FSH levels, 6) testicular unresponsiveness to hCG stimulation, and 7) no abnormal step up in testosterone biosynthesis precursors (194,204) (table 6).

Several different variants in the LHCGR gene were reported in patients with Leydig cell hypoplasia in both sexes (194,205).

Table 6. Phenotype of 46,XY Subjects with the Complete Form of Leydig Cell Hypoplasia
Inheritance	Autosomal recessive
External genitalia	Female, occasionally mild clitoromegaly or labial fusion
Müllerian derivatives	Absent
Wolffian ducts derivatives	Absent or vestigial
Testes	Inguinal or intra-abdominal, slightly subnormal size
Puberty	Absence of spontaneous virilization or feminization
Hormonal diagnosis	Elevated serum LH, normal or slightly elevated FSH and very low testosterone levels with normal levels of testosterone precursors
Gender role	Female
LHCGR gene location	2p21
Molecular defect	Pathogenic variants in LHCGR gene (complete inactivation) and in the internal exon 6A LHCGR(increase of nonfunctional isoform); defects in LHCGRwere not identified in several families
Treatment	Estrogen replacement at pubertal age, bilateral orchiectomy and vaginal dilation
Outcome	Female gender role and behavior, infertility

In contrast to the homogenous phenotype of the complete form of Leydig cell hypoplasia, the partial form features a broad spectrum, ranging from incomplete male sexual differentiation characterized by micropenis and/or hypospadias to hypergonadotropic hypogonadism without ambiguity of the male external genitalia (194,195,206,207). Testes are cryptorchidic or in the scrotum and during puberty, partial virilization occurs and testicular size is normal or only slightly reduced, while penile growth is significantly impaired. Spontaneous gynecomastia does not occur. Before puberty, the testosterone response to the hCG test is subnormal without accumulation of testosterone precursors. After puberty, LH levels are elevated as a result of insufficient negative feedback of gonadal steroid hormones on the anterior pituitary and testosterone levels are intermediate between those of children and normal males.

Several mutations in the LHCGR gene have also been identified in patients with the partial form of Leydig cell hypoplasia. Latronico et al. reported the first homozygous mutation in the LHCGR (p.Ser616Tyr) in a boy with micropenis (207). Subsequently, other milder mutations were identified in further patients with the partial form of Leydig cell hypoplasia (194,195,207). In vitro studies showed that cells transfected with LHCGR gene containing these mutations had an impaired hCG-stimulated cAMP production (195,207).

Leydig cell hypoplasia was found to be a genetic heterogenous disorder since Zenteno et al. (197) ruled out, by segregation analysis of a known polymorphism in exon 11 of the LHCG receptor gene, molecular defects in the LHCG receptor as being responsible for Leydig cell hypoplasia in three siblings with 46,XY DSD. Most inactivating mutations of the LHCGR are missense mutations that result in a single amino acid substitution in the LHCGR. In addition, mutations causing amino acid deletions, amino acid insertions, splice acceptor mutation or premature truncations of the receptor have also been reported (208). LHCGR mutations are usually located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction.

Although it is well known that hCG and LH act by stimulating a common receptor, a differential action of them in the LHCGR has been suggested. The identification of a deletion of exon 10 of the LHCGR in a patient with normal male genitalia at birth, but no pubertal development indicated that the mutant LHCGR was responsive to fetal hCG, but resistant to pituitary LH. The binding affinity of hCG for LHCGR was normal in vitro analysis, suggesting that exon 10 is necessary for LH, but not for hCG action (199).

The identification and characterization of a novel, primate-specific bona fide exon (exon 6A) within the LHCGR determined a new regulatory element within the genomic organization of this receptor and a new potential mechanism of this disorder. Kossack et al analyzing the exon 6A in 16 patients with 46,XY DSD due to Leydig cells hypoplasia without molecular diagnosis, detected mutations (p.A557C or p.G558C) in three patients. Functional studies revealed a dramatic increase in expression of the mutated internal exon 6A transcripts, resulting in the generation of predominantly nonfunctional isoforms of the LHCGR, thereby preventing its proper expression and functioning (209).

A new compound heterozygous mutation of the LHCGR, constituted by a previously described missense mutation (p.Cys13Arg) and a large deletion of the paternal chromosome 2 was identified by array-Comparative Genomic Hybridization (array-CGH) in a 46,XY infant with sexual ambiguity and low hCG-stimulated testosterone levels associated with high LH and FSH levels (200).

In addition, causative mutations in LHCGR were absent in around 50% of the patients strongly suspected to have Leydig cell hypoplasia. These findings supported the idea that other genes must be implicated in the molecular basis of this disorder.

We observed that 46,XX sisters of the patients with 46,XY DSD due to Leydig cell hypoplasia, carrying the same homozygous mutation in the LHCGR, have primary or secondary amenorrhea, spontaneous breast development, infertility, normal or enlarged cystic ovaries with elevated LH and LH/FSH ratio, normal estradiol and progesterone levels for early to mid-follicular phase, but not for luteal phase levels, confirming lack of ovulation (198,207,210). Our findings were subsequently confirmed by other authors who studied 46,XX sisters of 46,XY DSD patients with Leydig cell hypoplasia (201,202,211).

Subsequently, a novel homozygous missense mutation, p.N400S, has been identified by whole genome sequencing in two sisters with empty follicle syndrome (204).

Table 7. Phenotype of 46,XY Subjects with Partial Leydig Cells Hypoplasia
Inheritance	Autosomal recessive
External genitalia	Atypical to male
Müllerian derivatives	Absent
Wolffian ducts derivatives	Rudimentary to male
Testes	Scrotum, labial folds, or inguinal regions, normal or only slightly subnormal size
Puberty	Partial virilization without gynecomastia, discrepancy between reduced penis size and normal testicular growth
Hormonal diagnosis	Elevated serum LH levels, normal or slightly elevated FSH and low T levels with normal levels of T precursors in relation to T
Gender role	Male
LHCGR gene location	2p21
Molecular defect	Variants which confer partial inactivation of LHCGR
Treatment	Repair of the hypospadias, testosterone replacement at pubertal age
Outcome	Male gender role and behavior, possible fertility under treatment

46,XY DSD Due to Enzymatic Defects in Testosterone Synthesis

Six enzymatic defects that alter the normal synthesis of testosterone have been described to date (Figure 10). Three of them are associated with defects in cortisol synthesis leading to congenital adrenal hyperplasia. All of them present an autosomal recessive mode of inheritance and genetic counseling is mandatory since the chance of recurring synthesis defects among siblings is 25%.

Figure 10. Standard steroidogenesis and alternative pathway to DHT synthesis.

DEFECTS IN ADRENAL AND TESTICULAR STEROIDOGENESIS

Adrenal hyperplasia syndromes are examples of hypoadrenocorticism or mixed hypo- and hyper cortico-adrenal steroid secretion. Synthesis of cortisol or both cortisol and aldosterone are impaired. When cortisol production is impaired, there is a compensatory increase in ACTH secretion. If mineralocorticoid production is impeded, there is a compensatory increase in renin-angiotensin production. These compensatory mechanisms may return cortisol or aldosterone production to normal or near normal levels, but at the expense of excessive production of precursors that can cause undesirable hormonal effects.

Lipoid Congenital Adrenal Hyperplasia due to Deficiency of the Steroidogenic Acute Regulatory Protein (StAR)

StAR is a mitochondrial phosphoprotein which facilitates the influx of cholesterol from the outer to the inner mitochondrial membrane for the subsequent action of the P450scc enzyme (212).

StAR is encoded by the STAR gene and its deficiency leads to congenital lipoid adrenal hyperplasia (CLAH), the most severe form of congenital adrenal hyperplasia (213) . Lipoid adrenal hyperplasia is rare in Europe and America, but it is thought to be the second most common form of adrenal hyperplasia in Japan where 1 in 300 individuals carries the p.Q258X variant (214).

Affected subjects are phenotypic females irrespective of gonadal sex or sometimes have slightly virilized external genitalia with or without cryptorchidism, underdeveloped internal male organs and an enlarged adrenal cortex, engorged with cholesterol and cholesterol esters (215). Adrenal steroidogenesis deficiency leads to salt wasting, hyponatremia, hyperkalemia, hypovolemia, acidosis, and death in infancy, although patients can survive to adulthood with appropriate mineralocorticoid- and glucocorticoid-replacement therapy (215).

Hormonal diagnosis is based on high ACTH and renin levels and the presence of low levels of all glucocorticoids, mineralocorticoids, and androgens.

The disease was firstly attributed to P450scc deficiency, but most of the cases studied through molecular analysis showed an intact P45011A gene and its RNA (216). Since StAR is also required for the conversion of cholesterol to pregnenolone, molecular studies were performed in StAR gene and variants were found in most of the affected patients (217) Congenital lipoid adrenal hyperplasia (LCAH) in most Palestinian cases is caused by a founder c.201_202delCT variant causing premature termination of the StAR protein (217) Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells since 1 yr. of age, positive placental alkaline phosphatase and octamer binding transcription factor (OCT4) staining indicating a neoplastic potential (217).

A two-hit model has been proposed by Bose et al. (216) as the pathophysiological explanation for LCAH. In response to a stimulus (e.g., ACTH), the normal steroidogenic cell recruits cholesterol from endogenous synthesis, stored lipid droplets or low-density lipoprotein-receptor mediated endocytosis.

Subsequently StAR promotes the cholesterol transport from the outer to the inner mitochondrial membrane in which cholesterol is further processed to pregnenolone. In cells with mutant StAR (first hit), there is no rapid steroid synthesis, but still some StAR-independent cholesterol flows into the mitochondria, resulting in a low level of steroidogenesis. Due to increased steroidogenic stimuli in response to inadequately low steroid levels, additional cholesterol accumulates. Massive cholesterol storage and resulting biochemical reactions eventually destroy all steroidogenic capacity (second hit) (217). This two-hit model has been confirmed by clinical studies (218) as well as StAR knockout mice research (219).

The human STAR gene is localized on chromosome 8p11.2 and consists of seven exons (220). It is translated as a 285-amino acid protein including a mitochondrial target sequence (N terminal 62 amino acids), which guides StAR to the outer mitochondrial membrane and a cholesterol binding site, which is located at the C-terminal region. In vitro studies revealed that StAR protein lacking the N terminal targeting sequence (N-62 StAR) can still stimulate steroidogenesis in transfected COS-1 cells, whereas variants in the C-terminal region led to severely diminished or absent function (221-223). Most of the STAR gene variants associated with LCAH are located in the C-terminal coding region between exons 5 and 7 StAR related lipid transfer (START) domain (224). Mild phenotype of lipoid CAH is a recognized disorder caused by StAR variants that retain partial activity (225). Affected males can present with adrenal insufficiency resembling autoimmune Addison disease with micropenis or normal development with hypergonadotropic hypogonadism (224,225). More than 40 StAR variants causing classic lipoid CAH have been described (217,226,227), but very few partial loss-of-function variants have been reported (224-226). Therefore, there is a broad clinical spectrum of StAR variants, however, the StAR activities in vitro correlate well with clinical phenotypes (228).

Three 46,XY patients with the homozygous p.R188C STAR variant causing primary adrenocortical insufficiency without atypical genitalia were reported (229). Patients with nonclassical lipoid CAH may present with male genitalia and preserved testicular function (230).

Table 8. Phenotype of 46,XY Subjects with StAR Deficiency
Inheritance	Autosomal recessive
External genitalia	Female Micropenis (mild form)
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Absent -> hypoplastic
Testes	Small size
Clinical Features	Early adrenal insufficiency; no pubertal development; hypergonadotropic hypogonadism
Hormonal diagnosis	Elevated ACTH and renin levels; low levels of all glucocorticoids, mineralocorticoids, and androgens
Gender role	Female Male (mild form)
STAR gene location	8p11.2
Molecular defect	Inactivating variants in STAR
Treatment	Early gluco- and mineralocorticoid replacement; estrogen replacement at pubertal age
Outcome	Infertile, female or male gender role and behavior

Deficiency of P450 Side Chain Cleavage Enzyme (P450scc) Due to Variants in CYP11A1

The first step in the conversion of cholesterol to hormonal steroids is hydroxylation at carbon 20, with subsequent cleavage of the 20-22 side chain to form pregnenolone. In steroidogenic tissues, such as adrenal cortex, testis, ovary, and placenta, this is the initial and rate-limiting step in steroidogenesis. This reaction, known as cholesterol side-chain cleavage, is catalyzed by a specific cytochrome P450 called P450scc or CYP11A1 encoded by the CYP11A1 gene (231).

A number of patients with CYP11A1 variants have now been described (232-235), including late-onset non-classical forms secondary to variants that retain partial enzyme activity (236,237). Clinically, these patients are indistinguishable from those with lipoid CAH, but none of them present enlarged adrenals that characterize lipoid CAH.

Analyzing infants with adrenal failure and disorder of sexual differentiation compound heterozygous variants in CYP11A1 have been identified, recognizing that this disorder may be more frequent than originally thought. The phenotypic spectrum of P450scc deficiency ranges from severe loss-of-function variants associated with prematurity, complete under androgenization, and severe early-onset adrenal failure, to partial deficiencies found in children born at term with mild masculinization and later-onset adrenal failure (236,237).

3β-Hydroxysteroid Dehydrogenase type II Deficiency

3β-hydroxysteroid dehydrogenase 2 (3βHSD2) deficiency is a rare form of congenital adrenal hyperplasia (CAH), with fewer than 200 cases reported in the world literature.

3β-HSD converts 3β-hydroxy 5 steroids to 3-keto 4 steroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex steroids Two forms of the enzyme have been described in man: the type I enzyme which is expressed in placenta and peripheral tissues such as the liver and skin, and type II that is the major form expressed in the adrenals and gonads (238). The two forms are very closely related in structure and substrate specificity, though the type I enzyme has higher substrate affinities and a 5-fold greater enzymatic activity than type II (238).

Male patients with 3β-HSD type II deficiency present with atypical external genitalia, characterized by microphallus, proximal hypospadias, bifid scrotum and a blind vaginal pouch associated or not with salt loss (239,240). Precocious pubarche and gynecomastia at pubertal stage is a common phenotype in 3β-HSD type II deficiency (241).

Serum levels of Δ-5 steroids such as pregnenolone, 17OHpregnenolone (17OHPreg), DHEA, DHEAS are elevated and basal levels of 17OHPreg and 17OHPreg/17OHP ratio are the best markers of this deficiency in both prepubertal and postpubertal stage. Δ-4 steroids are slightly increased due to the peripheral action of 3β-HSD type I enzyme but the ratio of Δ-5/Δ-4 steroids is elevated. Cortisol secretion is reduced but the response to exogenous ACTH stimulation varies from decreased (more severe deficiency) to normal. At adult age, affected males can reach normal or almost normal levels of testosterone due to the peripheral conversion of elevated Δ-5 steroids by 3β-HSD type I enzyme and also due to testicular stimulation by the high LH levels (242).

The human genome encodes two functional 3βHSD genes on chromosome 1p13.1. The HSD3B2 gene is expressed in adrenal and gonads and consists of four exons coding for a 372 amino acid protein (243). To date, around 40 variants in the HSD3B2 gene have been described. Most of them are base substitutions, and they are located especially at the N-terminal region of the protein. The amino acids A10, A82, P222 and T259 could be considered a hotspot since different variants were reported in these HSD3B2 positions.

Variants abolishing 3β-HSD type II activity lead to congenital adrenal hyperplasia (CAH) with severe salt-loss (244). Variants that reduce, but do not abolish type II activity ( > 5% of wild type 3βHSD2 activity in vitro) lead to CAH with mild or no salt-loss, which in males is associated with 46,XY DSD due to the reduction in androgen synthesis (241,242,245,246). Male subjects with 46,XY DSD due 3β-HSD type II deficiency without salt loss showed clinical features in common with the deficiencies of 17β-HSD3 and 5α-reductase 2.

Most of the patients were raised as males and kept the male social sex at puberty. In one Brazilian family, two cousins with 46,XY DSD due to 3β-HSD type II deficiency were reared as females; one of them was underwent orchiectomy in childhood and kept the female social sex; the other did not undergo orchiectomy at childhood and changed to male social sex at puberty (246).

There is little data on the outcomes of 3β-HSD type II deficiency. A mixed longitudinal and cross-sectional study from a single Algerian center reported 14 affected subjects (8 females) with pathogenic variants in HSD3B2 gene (247). Premature pubarche was observed in four patients (3F:1M). Six patients (5F:1M) entered puberty spontaneously, aged 11 (5-13) years in 5 girls and 11.5 years in one boy. Testicular adrenal rest tumors were found in three boys. Four girls reached menarche at 14.3 (11-14.5) years, with three developing adrenal masses and polycystic ovary syndrome (PCOS), with radiological evidence of ovarian adrenal rest tumor in one. The median IQ was 90 (43-105), >100 in only two patients and <70 in three of them (247).

Table 9. Phenotype of 46,XY Subjects with 3β-HSD Type II Deficiency
Inheritance	Autosomal recessive
External genitalia	Atypical (proximal hypospadias, bifid scrotum, urogenital sinus), precocious pubarche
Müllerian derivatives	Absent
Wolffian duct derivatives	Normal
Testes	Well developed; generally topic
Clinical features	Adrenal insufficiency or not in infancy; virilization at puberty with or without gynecomastia
Hormonal diagnosis	Elevated basal and ACTH-stimulated 17OHPreg and 17OHPreg/17OHP ratio
Gender role	Male; female to male
HSD3B2 gene location	1p13.1
Molecular defect	Inactivating variants in HSD3B2
Treatment	Glucocorticoid replacement along with mineralocorticoids in salt-losing form; at puberty variable necessity for testosterone replacement
Outcome	Variable spermatogenesis; fertility possible by in vitrofertilization

Combined 17-Hydroxylase and C-17-20 lyase deficiency

CYP17 is a steroidogenic enzyme that has dual functions: hydroxylation and lyase. It is located in the fasciculata and reticularis zone of the adrenal cortex and gonadal tissues. The first activity results in hydroxylation of pregnenolone and progesterone at the C(17) position to generate 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, while the second enzyme activity cleaves the C(17)-C(20) bond of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone to form dehydroepiandrosterone and androstenedione, respectively. The modulation of these two activities occurs through cytochrome b5, necessary for lyase activity (248).

Deficiency of adrenal 17-hydroxylation activity was first demonstrated by Biglieri et al. (249). The phenotype of 17-hydroxylase deficiency in most of the male patients described is a female-like or slightly virilized external genitalia with blind vaginal pouch, cryptorchidism and high blood pressure, usually associated with hypokalemia. New in 1970, reported the first affected patient with atypical genitalia which was assigned to the male sex (250). The 17-hydroxylase deficiency is the second most common cause of CAH in Brazil (251).

At puberty, patients usually present sparse axillary and pubic hair. Male internal genitalia are hypoplastic and gynecomastia can appear at puberty. Most of the male patients were reared as females and sought treatment due to primary amenorrhea or lack of breast development. Genetic female patients may also be affected and present normal development of internal and external genitalia at birth and hypergonadotropic hypogonadism and amenorrhea at post pubertal age; enlarged ovaries at adult age and infarction from twisting can occur (252,253). These patients do not present signs of glucocorticoid insufficiency, due to the elevated levels of corticosterone, which has a glucocorticoid effect. The phenotype is similar to 46,XX or 46,XY complete gonadal dysgenesis and the presence of systemic hypertension and absence of pubic hair in post pubertal patients suggests the diagnosis of 17-hydroxylase deficiency (254).

Serum levels of progesterone, corticosterone, and 18-OH-corticosterone are elevated, while aldosterone, 17-OH-progesterone, cortisol, androgens and estrogens are decreased. Martin et al, performed a clinical, hormonal, and molecular study of 11 patients from 6 Brazilian families with the combined 17-alpha-hydroxylase/17,20-lyase deficiency phenotype (255). All patients had elevated basal serum levels of progesterone and suppressed plasma renin activity. The authors concluded that basal progesterone measurement is a useful marker of P450c17 deficiency and suggest that its use should reduce the misdiagnosis of this deficiency in patients presenting with male DSD, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.

Excessive production of deoxycorticosterone and corticosterone results in systemic hypertension, suppression of renin levels and inhibition of aldosterone synthesis. The CYP17A1 gene, which encodes the enzymes 17-hydroxylase and 17-20 lyase, is a member of a gene family within the P450 supergene family and is mapped at 10q24.3 (254) (256). Several variants in the CYP17A1 gene have been identified in patients with both 17-hydroxylase and 17,20 lyase deficiencies (252,253,257). Four homozygous variants, p.A302P, p.K327del, p.E331del and p.R416H, were identified by direct sequencing of the CYP17A1 gene. Both P450c17 activities were abolished in all the mutant proteins but the mutant proteins were normally expressed, suggesting that the loss of enzymatic activity is not due to defects of synthesis, stability, or localization of P450c17 proteins (257).

Glucocorticoid replacement for hypertension management, gonadectomy and estrogen replacement at puberty for patients reared in the female social sex are indicated. In male patients, androgen replacement is usually necessary since they present very low levels of testosterone. These patients are very sensitive to glucocorticoids and low doses of dexamethasone (0.125-0.5 mg at night) are sufficient to control blood pressure. In some patients, however, estrogens might aggravate hypertension. The control of blood pressure can be initially achieved by salt restriction although mineralocorticoid antagonists might be necessary (257).

Table 10. Phenotype of 46,XY Subjects with 17a-Hydroxylase and 17,20-Lyase Deficiency
Inheritance	Autosomal recessive
External genitalia	Female like --> atypical
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Hypoplastic --> normal
Testes	Intra-abdominal or inguinal
Clinical features	Low renin hypertension; absent or slight virilization at puberty; gynecomastia
Hormonal diagnosis	Elevated progesterone, DOC, corticosterone; low plasma renin activity low cortisol not stimulated by ACTH
Gender role	Female in most patients
CYP17 gene location	10q24.3
Molecular defect	Variants in CYP17A1 gene
Treatment	Repair of sexual ambiguity; glucocorticoid and estrogen or testosterone replacement according to social sex
Outcome	Female behavior, infertility

Cytochrome P450 Reductase (POR) Deficiency (Electron Transfer Disruption)

The apparent combined P450C17 and P450C21 deficiency is a rare variant of congenital adrenal hyperplasia, first reported by Peterson et al in 1985 (258). Affected girls and boys are born with atypical genitalia, indicating intrauterine androgen excess in females and androgen deficiency in males. Boys and girls can also present with skeletal malformations, which in some cases resemble a pattern seen in patients with Antley-Bixler syndrome. Findings of biochemical investigations of urinary steroid excretion in affected patients have shown accumulation of steroid metabolites, indicating impaired C17 and C21 hydroxylation, suggesting concurrent partial deficiencies of the 2 steroidogenic enzymes, P450C17 and P450C21. However, sequencing of the genes encoding these enzymes showed no variants, suggesting a defect in a cofactor that interacts with both enzymes. POR is a flavoprotein that donates electrons to all microsomal P450 enzymes, including the steroidogenic enzymes P450c17, P450c21 and P450aro (259). Shephard et al. (1989) isolated and sequenced cDNA clones that encode the rat and human NADPH-dependent cytochrome P-450 reductase and located the human gene at 7q11.2 (260).

The underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency was defined in 3 patients, who were compound heterozygotes for variants in POR (259,260). Antley-Bixler syndrome is characterized by craniosynostosis, severe midface hypoplasia, proptosis, choanal atresia/stenosis, frontal bossing, dysplastic ears, depressed nasal bridge, radio-humeral synostosis, long bone fractures, femoral bowing, phalangeal malformation (arachno-/campto-/clinodactyly, brachy-tele-phalanges, rocker bottom feet) and urogenital abnormalities (259). The occurrence of genital abnormalities in patients with Antley-Bixler syndrome, especially females was reported in 2000 (261). In a recent large survey of patients with Antley-Bixler syndrome, it was demonstrated that individuals with an Antley-Bixler-like phenotype and normal steroidogenesis have FGFR2 variants, whereas those with atypical genitalia and altered steroidogenesis have POR deficiency (262). The skeletal malformations observed in many, but not all patients with POR deficiency, are thought to be due to disruption of enzymes involved in sterol synthesis, 14α-lanosterol demethylase (CYP51A1) and squalene epoxidase, and disruption of retinoic acid metabolism catalyzed by CYP26 isoenzymes that depend on electron transfer from POR (263).

Pubertal presentations in females with congenital POR deficiency were described. Incomplete pubertal development and large ovarian cysts prone to spontaneous rupture were the predominant findings in females (264).The ovarian cysts may be driven not only by high gonadotropins but possibly also by impaired CYP51A1-mediated production of meiosis-activating sterols due to mutant POR. In the two boys evaluated, pubertal development was more mildly affected, with some spontaneous progression. These findings may suggest that testicular steroidogenesis may be less dependent on POR than adrenal and ovarian steroidogenesis (265).

Table 11. Phenotype of 46,XY Patients with POR Deficiency
Inheritance	Autosomal recessive
External genitalia	Atypical
Müllerian duct derivatives	Normally developed
Wolffian duct derivatives	Normally developed
Testes	Well developed, frequent cryptorchidism
Hormonal diagnosis	Low T and cortisol and elevated basal ACTH, Prog and 17OHP
POR gene location	7q11.2
Molecular defect	Inactivating variants of POR gene
Puberty	Spontaneous pubertal development in males
Gender role	Male
Treatment	Repair of sexual ambiguity; glucocorticoid replacement and estrogen or testosterone replacement according to social sex
Outcome	Puberty development, fertility?

DEFECTS IN TESTICULAR STEROIDOGENESIS

Three defects in testosterone synthesis that are not associated with adrenal insufficiency have been described: CYP17A1 deficiency, cytochrome B5 deficiency and 17-β-HSD3 deficiency

CYP17A1 Deficiency

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. The existence of true isolated 17,20-lyase deficiency has been questioned because 17-α-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme and because combined deficiencies of both activities were found in functional studies of the variant found in a patient thought to have had isolated 17,20-lyase deficiency (266,267). Later, clear molecular evidence of the existence of isolated 17,20 desmolase deficiency was demonstrated (268).

The patients present atypical genitalia with micropenis, proximal hypospadias and cryptorchidism. Gynecomastia Tanner stage V can occur at puberty (268).

Elevated serum levels of 17-OHP and 17-OHPreg, with low levels of androstenedione, dehydroepiandrosterone and testosterone, are described. The hCG stimulation test results in a slight stimulation in androstenedione and testosterone secretion with an accumulation of 17-OHP and 17-OHPreg.

The CYP17A1 gene of two Brazilian 46,XY DSD patients with clinical and hormonal findings indicative of isolated 17,20-lyase deficiency, since they produce cortisol normally, were studied. Both were homozygous for missense variants in CYP17A1 (268). When expressed in COS-1 cells, the mutants retained 17α-hydroxylase activity and had minimal 17,20-lyase activity. Both variants alter the electrostatic charge distribution in the redox-partner binding site, so that the electron transfer for the 17,20-lyase reaction is selectively lost (268).

Table 12. Phenotype of 46,XY Subjects with 17,20 Lyase Deficiency
Inheritance	Autosomal recessive
External genitalia	Atypical (proximal hypospadias, bifid scrotum, urogenital sinus)
Müllerian derivatives	Absent
Wolffian ducts derivatives	Hypoplastic --> normal
Testes	In the inguinal region, small size
Clinical features	Gynecomastia variable; poor virilization at puberty
Hormonal diagnosis	Elevated 17OHP and 17OHP/A ratio after hCG stimulation and decreased A and T levels;
Gender role	Male or female
CYP17 gene location	10q24.3
Molecular defect	Variants in the redox partner binding site of CYP17A1 enzyme
Treatment	Repair of hypospadias and gynecomastia; testosterone replacement at pubertal age
Outcome	Male or female behavior

Cytochrome B5 deficiency (Allosteric Factor for P450c17 and POR Interaction)

In 1986, Hegesh et al described a 46,XY DSD patient with type IV hereditary methemoglobinemia (269). The patient had a 16-bp deletion in the cytochrome b5 mRNA leading to a new in-frame termination codon and a truncated protein. The etiology of 46,XY DSD in this patient was attributed to the cytochrome b5 defect since cytochrome b5 acts as an allosteric factor, promoting the interaction of. P450c17 and POR favoring 17,20 lyase reactions (270).

Two homozygous variants in CYB5 in 46,XY DSD patients with elevated methemoglobin levels but without clinical phenotype of methemoglobinemia were reported (269).

46,XY DSD due to 17β-HSD 3 Deficiency

This disorder consists of a defect in the last phase of steroidogenesis when androstenedione is converted to testosterone and estrone to estradiol. This disorder was described by Saez and his colleagues (271) and is the most common disorder of androgen synthesis, reported from several parts of the world (272,273).

There are 5 steroid 17β-HSD enzymes that catalyze this reaction (274) and 46,XY DSD results from variants in the gene encoding the 17β-HSD3 isoenzyme (275). Patients present female-like or atypical genitalia at birth, with the presence of a blind vaginal pouch, intra-abdominal or inguinal testes and epididymis, vasa deferentia, seminal vesicles and ejaculatory ducts. Most affected males are raised as females (276,277), but some have less severe defects in virilization and are raised as males (274). Virilization in subjects with 17β-HSD3 deficiency occurs at the time of expected puberty. This late virilization is usually a consequence of the presence of testosterone in the circulation because of the conversion of androstenedione to testosterone by some other 17β-HSD isoenzyme (presumably 17β-HSD 5) in extra-gonadal tissue and, occasionally, of the secretion of testosterone by the testes when levels of LH are elevated in subjects with some residual 17β-HSD3 function (274,277). However, the discrepancy between the failure of intrauterine masculinization and the virilization that occurs at the time of expected puberty is poorly understood. A limited capacity to convert androstenedione into testosterone in the fetal extragonadal tissues may explain the impairment of virilization of the external genitalia in the newborn. Bilateral orchiectomy resulted in a clear reduction of androstenedione levels indicating that the main origin of this androgen is the testis (278). 46,XY DSD phenotype is sufficiently variable in 17β-HSD3 deficiency to cause problems in accurate diagnosis, particularly in distinguishing it from partial androgen insensitivity syndrome (276,279).

Laboratory diagnosis is based on elevated serum levels of androstenedione and estrone and low levels of testosterone and estradiol resulting in elevated androstenedione/testosterone and estrone/ estradiol ratios or low (or low testosterone/androstenedione and estradiol/estrone ratios) indicating impairment in the conversion of 17-keto into 17-hydroxysteroids. Testosterone/Androstenedione ratio of 0.4±0.2 was found in prepubertal patients with 17β-HSD3 deficiency after hCG stimulation. Based on these data, a T/A ratio below <0.8 is suggestive of 17β-HSD3 deficiency (272). At the time of expected puberty, serum LH and testosterone levels rise in all affected males and testosterone levels may reach the normal adult male range (277,278).

Pitfalls in the hormonal diagnosis of 17β-HSD3 deficiency had been reported in the literature. Two of the fourteen cases of 17β-HSD3 deficiency reported from the UK database had a T/A ratio > 0.8 (276). Both patients were from a consanguineous pedigree, with two affected sisters (both assigned in the female gender) and one nephew. The former patient had atypical genitalia with proximal hypospadias and was assigned as male. The hCG test was performed at 2 years and 2 months of age, respectively, resulting in a T/A ratio of 3.4 and 1.5. Two other patients with atypical genitalia, who were also assigned in the female social sex, were evaluated at 5 months and 9.2 year of age, respectively (280). After the hCG stimulation test, there was a clear elevation of serum testosterone (measured by HPLC tandem mass spectrometry) with a small increase of the androstenedione levels resulting in a high T/A ratio (2.47 and 2.27 respectively). Sequencing of the HSD17B3 gene identified deleterious molecular defects in both alleles in both patients. The possible explanation for the normal T/A ratio in these 4 children is the individual and temporal variability in the HSD17B isoenzymes activity (280).

The disorder is due to homozygous or compound heterozygous variants in the HSD17B3 gene which encodes the 17β-HSD3 isoenzyme. Up to now, almost 40 variants in the HSD17B3 gene have been reported. These include missense, nonsense, exonic deletion, duplication and intronic splice site variants (274,277). Although allelic variants have been described throughout HSD17B3, a variant cluster region was identified in the exon 9. The 17β-HSD3 activity was completely eliminated in the majority of the HSD17B3 variants (276). Outside exon 9, the most frequent site of variant in HSD17B3 gene is the R80 in exon 3, which primarily disrupts the binding of the NADPH cofactor to the protein. The p.R80Q variant has been found in Palestinian, Brazilian, and Turkish families (281).

Most patients are raised as girls during childhood. Change to male gender role behavior at puberty has been frequently described in individuals with this disorder who were reared as females (282-285), including members of a large consanguineous family in the Gaza strip (286). In a review of all adult patients with 46,XY DSD due to 17β-HSD3 deficiency reared as female and not castrated during childhood reported until now, we found that 30 of them (61%) kept the female gender and 19 of them (39%) changed to male gender (277).

After a histological analysis of testicular tissue stained with hematoxylin-eosin from 40 reported cases of 46,XY patients with 17β-HSD3 deficiency, the prevalence of germ cell tumor was 5%, which is lower than the estimated GCT risk for some 46,XY DSD etiologies (287-289). However, the maintenance of the testes in male patients is safe if the testes can be positioned into the scrotum (277,290).

Table 13. Phenotype of 46,XY Patients with 17β-HSD 3 Deficiency
Inheritance	Autosomal recessive
External genitalia	Atypical, frequently female-like at birth
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Normally developed
Testes	Well developed, frequent cryptorchidism
Hormonal diagnosis	Low T and elevated basal and hCG-stimulated A and A/T ratio
HSD17B3 gene location	9q22
Molecular defect	Inactivating variants of HSD17B3
Puberty	Virilization at puberty; variable gynecomastia
Gender role	Most patients keep the female social sex; some change to male social sex
Treatment	Repair of sexual ambiguity; estrogen or testosterone replacement according to social sex
Outcome	Male or female gender identity; in males’ fertility possible by in vitro fertilization

ALTERNATIVE PATHWAY TO DHT SYNTHESIS

46,XY DSD Due to 3α-Hydroxysteroid Dehydrogenase Deficiency (AKR1C2 and AKR1C4 Defects)

Back in 1972, the molecular analysis of 46,XY DSD due to isolated 17,20-lyase deficiency patients failed to find variants in the CYP17A1 (248). However, the hormonal data was inconsistent with other adrenal enzymatic deficiencies. Therefore, the alternative or backdoor pathway was considered to explain the etiology of the DSD in these patients. The backdoor pathway was firstly described in marsupials and is remarkable for having both reductive and oxidative 3α-HSD steps: the reductive reaction converts 17-OH-dihydroprogesterone (17OH-DHP) into 17OH-allopregnanolone (17OH-Allo), and the oxidative reaction converts androstanediol into DHT (291,292) (Figure 6). Therefore, synthesis of dihydrotestosterone (DHT) occurs without the intermediacy of DHEA, androstenedione or testosterone (291). All the human genes participating in the backdoor pathway have not been identified, however it has been thought that the reductive 3α-HSD activity can be catalyzed by an aldo-keto reductase called AKR1C2 (293), as well as by other enzyme, such as the oxidative 3α-HSD activity by 17β-HSD6, also called as RoDH (294)and possibly by AKR1C4 (295).

The first reported cases with isolated 17,20 lyase deficiency from 1972 (266) were found to carry variants in two aldo-keto reductases, AKR1C2 and AKR1C4 which catalyze 3α-hydroxysteroid dehydrogenase activity. The two affected 46,XY females were compound heterozygotes for AKR1C2 variants, the p.I79V/H90Q and p.I79V/N300T. However, the mutant AKR1C2 enzymes retained 22-82% of wild-type activity in vitro analysis suggesting that another gene might be involved (248). Analysis of AKR1C cDNA found that AKR1C4 was spliced incorrectly and gene sequencing displayed an intronic variant 106 bases upstream from exon 2 that caused this exon skipping. So, in this family, a digenetic inheritance was found to impair testicular synthesis of DHT during prenatal life (296).

AKR1C2 is abundantly expressed in the fetal testis, but minimally expressed in the adult testis; on the other hand, the AKR1C4 was found in fetal and adult testes at lower levels (293). Therefore, it appears that both AKR1C2 and AKR1C4 participate in the backdoor pathway to DHT in the fetal testis, and that molecular defects in these genes appear to cause incomplete male genital development (297). However, the relative roles of these two AKR1C enzymes remain unclear and testosterone levels at adult age are not available in these patients (298).

All findings described above, which substantially advanced our understanding of the underlying mechanisms of male sexual differentiation, illustrate the importance of detailed studies of rare 17,20 lyase deficiency patients.

46,XY DSD DUE TO DEFECTS IN TESTOSTERONE METABOLISM

5α-Reductase Type 2 Deficiency

A condition named pseudo-vaginal perineo-scrotal hypospadias in 46,XY individuals was reported in 1961, in which the phenotype included female-like external genitalia, bilateral testes, and male urogenital tracts with a blind-ending vagina (299). Thereafter, experimental studies showed that the male external genitalia virilization depended on the conversion of testosterone into dihydrotestosterone (DHT), an enzymatic reaction catalyzed by the 5α-reductase enzyme. Further, that enzymatic deficiency was biochemically and clinically reported in 24 individuals from the Dominican Republic and two siblings from North America (300,301). Typically, affected individuals are born with female-like external genitalia but develop clinical and psychological virilization at puberty with no gynecomastia (300). Both studies characterized this syndrome as a genetic condition with an autosomal recessive pattern of inheritance, resulting from the inability to convert testosterone into DHT. Later, two different genes encoding two 5α-reductase isoenzymes were isolated by cloning technology: the 5α-reductase type 1 and 2 (SRD5A1 and SRD5A2) (302). Allelic variants in the SRD5A2 gene were found in two individuals from Papua New Guinea with clinical features of 5α-reductase type 2 deficiency, whereas controls did not have variants in this gene, suggesting that variants in the SRD5A2 were the molecular basis of this condition (303). Further, the SRD5A2 gene was mapped at chromosome 2 (2p23), containing 5 exons and 4 introns, and encoding a 254 amino-acids protein (304).

Since then, several SRD5A2 allelic variants have been reported across the whole gene in individuals presenting this particular 46,XY DSD (305). We recently reviewed all 5α-reductase type 2 deficiency cases reported in the literature. We identified 451 cases of 5α-reductase type 2 deficiency from several countries, harboring 121 different SRD5A2 allelic variants (306). These variants have been reported in all exons of this gene, but mainly are located at exons 1 (33%) and at exon 4 (25%). Among the 254 amino acids that make up the SR5A2 protein, we found allelic variants in the SRD5A2 gene in 76 of them (306).

Regarding the SRD5A2 allelic variants, most are missense variants, but small deletions, variants at splicing sites, stop codons, small indels (n = 20) and large deletions have also been described. We also identified homozygosity in 70% of the SRD5A2 allelic variants causing 5α-reductase type 2 deficiency (306).

Neonatal diagnosis was carried out in 29.7%, whereas the remaining had the 5α-reductase type 2 deficiency diagnosis later in life. Most cases were assigned as female (69.4%), and an association between higher scores of external genitalia virilization (less virilization) and female sex assignment was identified. However, when we divided the cases into those who were diagnosed after and before 1999, the percentage of male sex assignment rose from 26.8% to 42.8%, suggesting a temporal trend pointing toward an increased likelihood of 5α-reductase type 2 deficiency patients being raised as boys (305).

Intriguingly, 5α-reductase type 2 deficiency is a condition with no genotype-phenotype correlation (307-309). This observation is based on several 5α-reductase type 2 deficiency families carrying the same genotype but presenting a broad range of external genitalia virilization. However, some SRD5A2 variants are consistent in the way they affect phenotype. It is the case of the p.Arg246Gln variant, which is associated with more external genitalia virilization (304,310-312), and also the case of both p.Gly183Ser and p.Gln126Arg variants, that are consistently reported with more severe external genitalia under-virilization (304,313-315).

The diagnosis is usually made at birth, infancy or at puberty. In the newborn, the features of 46,XY DSD due to 5a-reductase type 2 deficiency overlap with other forms of male DSD such as androgen insensitivity syndrome (partial form) and testosterone synthesis defects (304,316).

At puberty or in young adult men, the basal hormonal evaluation demonstrates normal male serum testosterone levels, low or low/normal dihydrotestosterone levels, and elevated or normal serum testosterone to dihydrotestosterone ratio (307). For appropriated use of this ratio, the testosterone levels should be in the post puberal range. Likewise, in prepubertal children, a hCG stimulation to increase serum testosterone levels is necessary (313). The biggest challenge is the diagnosis in newborns. This difficult largely arises from the fact that even when serum testosterone has undergone a neonatal surge, the ratio of serum testosterone to dihydrotestosterone may be normal, because expression of the 5a-reductase type 1 enzyme can occasionally be higher than expected (317,318).

Measurement of dihydrotestosterone is difficult because this steroid is present in very low concentrations and has a high rate of cross-reactions (319). To obtain an accurate dihydrotestosterone measurement, a precise assay must be utilized since serum testosterone levels are higher than dihydrotestosterone levels (about 10-fold). Consequently, the separation of testosterone from dihydrotestosterone is necessary to provide and accurate dihydrotestosterone measurement. Using such methodologies, the testosterone/dihydrotestosterone ratio for 5a-reductase type 2 deficiency hormonal diagnosis is generally over 18 in most cases (320,321). However, the testosterone/dihydrotestosterone ratio for 5a-reductase type 2 deficiency hormonal diagnosis has been debatable.

Another approach to 5a-reductase type 2 deficiency diagnosis is the measurement of urinary steroids by gas chromatography– mass spectrometry (GC–MS) to determine the ratio of 5a- to 5ß- reduced steroids in urine. This evaluation is very helpful for the diagnosis in subjects at prepubertal age and in orchiectomized adults. In one review, extremely low ratios of 5a- to 5ß-reduced steroid metabolites in urine were pathognomonic for 5a-reductase type 2 deficiency (322). Based on the challenges on the hormonal diagnosis, genetic analysis of the SRD5A2 gene is recommended to confirm the diagnosis [39,40].

The management in subjects with female social sex includes a careful psychological evaluation of gender identity (323). Subsequent management is similar to that in women with other forms of 46,XY DSD (324). Treatment must simulate a normal puberty pattern and low to normal estrogen doses, considering the height, and it should be administered at the age of expected puberty (10 – 12 years old). After complete breast development, adult estrogen doses are maintained continuously. Progesterone replacement is not necessary because these patients do not have a uterus (11). For women with this condition, feminizing genitoplasty is often necessary to provide an adequate vaginal opening, a functional vaginal introitus, full separation between urethral and vaginal orifice and phallic erectile tissue removal (11). Vaginal dilatation to promote vaginal length with acrylic molds is recommended when the patients decide to initiate sexual activity (325). Laparoscopic orchiectomy is recommended for all female patients to avoid virilization and gonadal malignancies. Usually, testosterone replacement is not often necessary for male patients since most retain testes and present adequate testicular function towards puberty (307). However, since the degree of virilization is usually unsatisfactory in male patients, a limited use of intramuscular testosterone or transdermal dihydrotestosterone may be helpful to improve virilization (307,319). Maximum penile length is obtained after 6 months of high dose testosterone therapy (e.g., 500 mg of testosterone cypionate per week) (319). The therapeutic penile response does not result in normal penile length in all individuals, even when initiated during childhood, and the final penile length is still below -2 SD in most patients (326). Surgical treatment consists of orthophalloplasty, scrotoplasty, resection of the vaginal pouch and proximal and distal urethroplasty. Correction of hypospadias is indicated in early childhood (up to two years old) (326).

Gender change (from female to male) is common among 5α-reductase type 2 deficiency individuals (327). It occurs in over 50% among those assigned as girls in some series (328). It may change since there is growing evidence suggesting male sex assignment for 5α-reductase type 2 deficiency newborns to avoid gender incongruence and gender dysphoria (329-331).

Regarding long-term follow up in the males from the Sao Paulo cohort, most of these subjects were satisfied with the appearance of the external genitalia and sexual life, although a small penile length made sexual intercourse difficult for some of them (326). Most of the adult males patients get married, and those reared as male report a more satisfactory quality of life than the female patients (332). Among female individuals, most describe a satisfactory sexual life, but none are married or have adopted children (333).

Table 14. Phenotype of 46,XY Subjects with 5α-Reductase 2 Deficiency
Inheritance	Autosomal recessive
External genitalia	Atypical, small phallus, proximal hypospadias, bifid scrotum, blind vaginal pouch
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Normal
Testes	Normal size at inguinal, intra-abdominal region or topic
Puberty	Virilization at puberty, absence of gynecomastia
Hormonal diagnosis	Increased T/DHT ratio in basal and hCG-stimulation conditions in postpubertal patients and after hCG-stimulation in pre-pubertal subjects. Elevated 5β/5α C₂₁ and C₁₉ steroids in urine in all ages
SRD5A2 gene location	2 p23
Molecular defect	Inactivating variants in 5RD5A2
Gender role	Female → male in 60% of the cases
Treatment	High doses of T and/or DHT for 6 months to increase penis size
Outcome	Maximum penis size in males after treatment = 9 cm; fertility is possible by in vitro fertilization

46,XY DSD DUE TO DEFECTS IN ANDROGEN ACTION

Androgen Insensitivity Syndrome

Androgen insensitivity syndrome (AIS) is the most frequent etiology of 46,XY DSD individuals (334). The underlying molecular basis of AIS is variants in the androgen receptor gene (AR), which is located on the long arm of the X chromosome (Xq11-12) (335). AIS is an X-linked inherited condition, and up to 30% of AIS cases present de novovariants (334,336). Due to disruptive variants in the AR gene, affected individuals present a broad spectrum of under-virilization, which will depend upon the residual activity of the mutant AR. There are three phenotypes of AIS: complete (typically female external genitalia; CAIS), partial (a wide spectrum of external genitalia under virilization; PAIS) or mild (typically male external genitalia with further gynecomastia and/or infertility; MAIS) (337,338).

The AR contains eight exons and encodes a 920 amino-acids protein (10). The AR is composed of three major functional domains: the N-terminal transactivation domain (NTD), a central DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD), and a hinge region connecting the DBD and LBD (339,340). The main difference between the AR and other steroid receptors is the presence of a longer NTD (341). Exon 1 encodes for the NTD, while exons 2 and 3 encode for the DBD and exons 4-8 encode for the LDB (342). In the presence of androgens, the AR recruits multiple epigenetic coregulators. These co-regulators can be either co-activators or co-repressors and actupon AR influencing DNA binding, nuclear translocation, chromatin remodeling, AR stability and bridging AR with transcriptional machinery (343,344). AR coding region has two polymorphic trinucleotide repeat regions, located at exon 1, the CAG and GGC repeats (345). The length of these repeats can cause human diseases. In general, longer CAG repeats may lead to AR transactivation impairment whereas shorter CAG repeats may enhance the ARtransactivation (346). A high number of CAG repeats (>38) is the molecular cause of Spinal and Bulbar Muscular Atrophy (Kennedy’s disease) (347). This condition is characterized by severe muscular atrophy and a mild AISphenotype, including gynecomastia. On the other hand, shorter CAG repeats are related with increased risk for prostate cancer (348).

There are more than 800 variants in the AR gene reported in AIS patients (www.androgendb.mcgill.ca/; HGMD). Most of them are missense variants leading to amino acid substitutions (349). However, small indels, variants at splicing sites, premature stop codons and large deletions were also reported, most of them related to CAIS (350). Despite a well-characterized monogenic condition, AR variants are identified in 90-95% of CAIS, but only in 28-50% of PAIS (334,350). Therefore, the molecular diagnosis of PAIS individuals remains challenging. Advances in molecular biology have been helpful to clarify unusual molecular mechanisms or deep DNA alterations related to AIS. Alterations immediately upstream of the AR were identified in AIS patients without variants in the coding region of the AR, either by promoting aberrant AR transcripts, or disrupting AR expression by the insertion of a large portion of a long-interspersed element retrotransposon, which were proven to cause AIS (351,352). Rare synonymous variants within the encoding region of the AR gene were proven to play a role in AIS by disrupting splicing (353). The sequencing of intronic regions of the AR was able to identify a deep intronic variant leading to pseudo-exon activation in AIS (354). Additionally, studies involving AR variant-negative individuals with AIS revealed the deficiency of the androgen-responsive apolipoprotein D, indicating functional AIS, and epigenetic repression of the AR transcription was reported in a group of AIS variant-negative individuals, a condition defined as AIS type II (355). However, a specific role of certain coregulators in the pathophysiology of AIS is not established yet and the contribution of AR-associated coregulators in AIS remains poorly understood.

COMPLETE ANDROGEN INSENSITIVITY SYNDROME

Prenatal diagnosis of CAIS is possible and can be suspected based on the discordance between 46,XY karyotype on prenatal fetal sex determination and the identification of a female genitalia at prenatal ultrasound (356). At birth, CAIS individuals present typically female external genitalia. In childhood, the identification of an inguinal hernia in a girl may be a clinical indication of CAIS, since inguinal hernias in girls are rare (357).

At puberty, CAIS patients present with complete breast development and primary amenorrhea (358). Pubic hair and axillar hair are sparse in most of them, and Mullerian ducts are often absent in CAIS patients (337).

The endocrine evaluation after puberty shows normal or elevated serum testosterone levels and slightly elevated LH levels, whereas FSH levels can be slightly elevated, with normal presence of testosterone precursors (334).

Patients with CAIS are assigned and raised as girls and usually present a female gender identity (328,331). Estrogen replacement is recommended to induce puberty if bilateral gonadectomy has been performed before puberty. There is gonadal tumor risk in CAIS patients, but this risk is very low before puberty (359). Therefore, gonadectomy can be postponed because after puberty is complete in CAIS patients (360,361) . An increasing number of adult women with CAIS prefer to decline or delay gonadectomy for several reasons, such as fear of surgery, to avoid estrogen replacement, and expectations for future fertility (362).

Table 15. Phenotype of 46,XY Subjects with Complete Androgen Insensitivity Syndrome
Inheritance	X-linked recessive
External genitalia	Female
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Absent or vestigial
Testes	Inguinal or intra-abdominal, slightly subnormal size
Puberty	Complete breast development
Hormonal diagnosis	High or normal serum LH and T levels, normal or slightly elevated FSH levels
Gender role	Female
AR gene location	Xq11-12
Molecular defect	Pathogenic allelic variants in AR gene
Treatment	Psychological support Estrogen replacement after gonadectomy. Vaginal dilation for sexual intercourse
Outcome	Female identity, infertility

PARTIAL ANDROGEN INSENSITIVITY SYNDROME

Patients with PAIS have a broad spectrum of virilization impairment (337). The external genitalia ranged from predominantly female with clitoromegaly and labial fusion to predominantly male with micropenis and hypospadias. Testes are in the inguinal canal or labioscrotal folds or, less frequently, intraabdominal. At puberty, under-virilization and gynecomastia are observed (334). The final height of PAIS individuals is intermediate between the average height for control males and females. In addition, PAIS individuals presented decreased bone mineral density in the lumbar spine compared to controls (363). In male PAIS, gynecomastia is common at puberty which is helpful in the differential diagnosis from other 46,XY DSD etiologies (364).

In the endocrine analysis, serum LH levels are in the normal upper range or slightly elevated, and testosterone levels are normal or slightly elevated (334). A definitive diagnosis of PAIS is established by identifying a variant in the ARgene, but AR variants are found only in about 40% of PAIS (350).

The sex of rearing is female in half of the cases, and gender change is uncommon in PAIS patients either raised as female or male (328).

Estrogen replacement is necessary for female patients to induce adequate puberty since most female PAIS patients undergo gonadectomy in childhood (334,365). For male patients, androgen replacement, either to induce puberty or to enhance virilization post-puberty, is commonly required (11). High doses of intramuscular testosterone preparations or topical DHT can be tried for six months to improve virilization, but it is unnecessary after that (11).

If the testes are at the scrotum, gonadectomy is unnecessary in male PAIS individuals. However, bilateral gonadectomy is still recommended for female PAIS due to avoid partial virilization and due to the gonadal malignancy risk (288,337).

Table 16. Phenotype of 46,XY Subjects with Partial Androgen Insensitivity Syndrome
Inheritance	X-linked recessive
External genitalia	Broad spectrum from female with mild clitoromegaly to male with micropenis and/or hypospadias
Müllerian duct derivatives	Absent
Wolffian duct derivatives	Broad spectrum from absent or male
Testes	Eutopic, inguinal or intra-abdominal, normal or slightly subnormal size
Puberty	Gynecomastia
Hormonal diagnosis	High or normal serum LH and T levels, normal or slightly elevated FSH levels
Gender role	Female or male
AR gene location	Xq11-12
Molecular defect	Pathogenic variants in AR gene
Treatment	Females: surgical feminization, gonadectomy, replacement with estrogens at the time of puberty, vaginal dilation (if necessary) Males: hypospadias repair, bifid scrotum; high doses of T or DHT to increase penis size
Outcome	Infertility, female or male gender role

46,XY DSD DUE TO PERSISTENT MÜLLERIAN DUCT

Defect in AMH Synthesis or AMH Receptor

The development of female internal genitalia in a male individual is due to the incapacity of Sertoli cells to synthesize or secrete anti-Mullerian hormone (AMH) or to alterations in the hormone receptor. Persistent Müllerian duct syndrome (PMDS) phenotype can be produced by a variant in the gene encoding anti-Müllerian hormone or by avariant in the AMH receptor. These two forms result in the same phenotype and are referred to as type I and type II, respectively (366).

AMH is a 145,000 MW glycoprotein homodimer produced by Sertoli cells not only during the period when it is responsible for regression of the Müllerian ducts but also in late pregnancy, after birth, and even, albeit at a reduced rate, in adulthood (13,367,368).

AMH is a small gene containing 5 exons, located in chromosome19p.13.3 (367) and its protein product acts through its specific receptor type 2 (AMHR2) a serine/threonine kinase, member of the family of type II receptors for TGF-β-related proteins (369).

Affected patients present a male phenotype, usually along with bilateral cryptorchidism and inguinal hernia (368). Leydig cell function is preserved, but azoospermia is common due to the malformation of ductus deferens or agenesis of epididymis. When the hernia is surgically corrected, the presence of a uterus, fallopian tubes and the superior part of the vagina can be verified.

PMDS is a heterogeneous disorder that is inherited in a sex-limited autosomal recessive manner. variants in AMHgene or AMH receptor 2 gene in similar proportions are the cause of approximately 85% of the cases of PMDS (370,371). In the remaining cases the cause of the persistent Mullerian duct syndrome is unknown (368).

Normally, AMH levels are measurable during childhood and decrease at puberty. Patients with AMH gene defects have low AMH levels since birth whereas patients with variants in AMH receptor gene have elevated AMH levels (372).

Treatment is directed toward an attempt to assure fertility in males. Early orchiopexy, proximal salpingectomy (preserving the epididymis), and a complete hysterectomy with dissection of the vas deferens from the lateral walls of the uterus are indicated (368,373).

CONGENITAL NON-GENETIC 46,XY DSD

Maternal Intake of Endocrine Disruptors

The use of synthetic progesterone or its analogs during the gestational period has been implicated in the etiology of 46,XY DSD (374). Some hypotheses have been proposed to explain the effect of progesterone in the development of male external genitalia, such as reduction of testosterone synthesis by the fetal testes or a decrease in the conversion of testosterone to DHT due to competition with progesterone (also a substrate for 5α-reductase 2 action). The effect of estrogen use during gestation in the etiology of 46,XY DSD has not been confirmed to date (375). Recently, a study in Japanese subjects supports the hypothesis that homozygosis for the specific estrogen receptor alpha 'AGATA' haplotype may increase the susceptibility to the development of male genital abnormalities in response to estrogenic effects of environmental endocrine disruptors (376).

Environmental chemicals that display anti-androgenic activity via multiple mechanisms of action have been identified. They are pesticides, fungicides, insecticides, plasticizers and herbicides. They can work as androgen receptor antagonists like pesticides, or they can decrease mRNA expression of key steroidogenic enzymes and also the peptide hormone insl3 from the fetal Leydig cells, like plasticizers and fungicides (377).

Daily exposure to residues of a fungicide (vinclozolin), either alone or in association with a phytoestrogen genistein (present in soy products), induce hypospadias in 41% of mice, supporting the idea that exposure to environmental endocrine disruptors during gestation could contribute to the development of hypospadias (378).

Supporting the idea that exposure to a mixture of chemicals can produce greater incidences of genital malformations, Rider et al examined the effects of exposure to a mixture of two chemicals that act as androgen receptor antagonists. They observed that the exposure to vinclozolin (fungicide) alone resulted in a 10% incidence of hypospadias and no vaginal pouch development in male rats, whereas procymidone, another fungicide exposure, failed to generate malformations. However, the combined exposure resulted in a 96% incidence of hypospadias and 54% incidence of vaginal pouch in treated animals. Similar results were observed in phthalate (plasticizer) mixture studies (377).

Given that severe alterations of sexual differentiation can be produced in animal laboratory studies, the question arises of what would be expected in exposed humans given that humans are exposed to mixtures of compounds in their environment.

Congenital Non-Genetic 46,XY DSD Associated With Impaired Prenatal Growth

Despite the multiple genetic causes of 46,XY DSD, around 30-40% of cases remain without diagnosis. Currently, there is a frequent, non-genetic variant of 46,XY DSD characterized by reduced prenatal growth and lack of evidence for any associated malformation or endocrinopathy (379,380). Using the model of monozygotic twins, hypospadias has now been linked to low birth weight (379). We have identified a pair of 46,XY monozygotic twins (identical for 13 informative DNA loci) born at term after an uneventful pregnancy sustained by one placenta who were discordant for genital development (perineal hypospadias versus normal male genitalia) and postnatal growth (low birth weight versus normal birth weight). No evidence for uniparental disomy was found (381). The most plausible cause of incomplete male differentiation associated with early-onset growth failure is a post-zygotic, micro-environmental factor since different DNA methylation patterns associated with silencing of genes important for sex differentiation has been shown (382).

Additionally, three cohorts of undetermined 46,XY DSD report around 30% of cases as associated with low birth weight, indicating that adverse events in early pregnancy are frequent causes of congenital non-genetic 46,XY DSD (383-385).

A genetic defect that clarifies the etiology of hypospadias was not found in 41 non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition (385).

46,XY OVOTESTICULAR DSD

There are rare descriptions of 46,XY DSD patients with well characterized ovarian tissue with primordial follicles and testicular tissue, a condition that is histologically characterized 46,XY ovo-testicular DSD (386). The differential diagnosis of 46,XY ovo-testicular DSD with partial 46,XY gonadal dysgenesis should be performed considering that in the latter condition there are descriptions of dysgenetic testes with disorganized seminiferous tubules and ovarian stroma with occasional primordial follicles in the first years of life (46). To our knowledge there are no descriptions of an adult patient with 46,XY ovo-testicular DSD with functioning ovarian tissue, as occurs in all 46,XX ovo-testicular DSD. Therefore, the diagnosis of 46,XY ovo-testicular DSD is debatable.

NON-CLASSIFIED FORMS

Hypospadias

Hypospadias is one of the most frequent genital malformations in the male newborn and 40% of the cases are associated with other defects of the urogenital system (387). Hypospadias results from an abnormal penile and urethral development that appears to be a consequence of various mechanisms including genetic and environmental factors. It is usually a sporadic phenomenon, but familial cases can be observed, with several affected members (388,389).

The presence of hypospadias indicates an intra uterus interference in the correct genetic program of the complex tissue interactions and hormonal action through enzymatic activities or transduction signals. MAMLD1 (mastermind-like domain containing 1) has been reported to be a causative gene for hypospadias (390). It appears to play a supportive role in testosterone production around the critical period for sex development. To date, microdeletions involving MAMLD1 and nonsense and frameshift variants in the gene have been found in 46, XY DSD patients, suggesting that MAMLD1 variants cause 46,XY DSD primarily because of compromised fetal testosterone production, however, its role in the molecular network involved in fetal testosterone production is not known so far (391).

The activating transcription factor 3 (ATF3) expression was identified in the developing male urethra. Apparently ATF3 variants may influence the risk of hypospadias (392).

By definition, hypospadias is a form of 46,XY DSD and although most of the patients maintain fertility and masculinization at puberty, their testicular function should be assessed to rule out causes such as defects in testosterone synthesis and action, which require hormonal treatment and genetic counseling in addition to surgical treatment.

GONADAL TUMOR DEVELOPMENT IN 46,XY DSD PATIENTS

Any disturbance in the gonadal development, including testicular descent, increases the risk of developing gonadal malignancies (288). For inherited disturbances in gonadal development or endocrine alterations, patients with 46,XY DSD are at increased risk of developing type II germ cell tumors (GCT) (289). In testicular tissues, GCTs comprise both premalignant conditions, such as germ cell neoplasia in situ (GCNIS) and malignant invasive germ cell tumors, including seminomas and non-seminomas (393).

The term GCNIS was introduced in the 2016 WHO classification of urological tumors to define precursor lesions of invasive GCTs, since GCNIS has the potential to develop into several types of GCTs (394,395). GCNIS cells are fetal gonocytes present in the seminiferous tubules arrested during gonadal development that failed to mature into spermatogonia (396). GCNIS are often detected in testicular tissues from 46,XY DSD subjects (397). It is estimated that 50% of GCNIS progress to an invasive GCT in five years (396,398,399).

A high risk of GCT is found when sex determination is disrupted at an early stage of Sertoli cell differentiation (due to abnormalities in SRY, WT1, SOX9) (289,397,400). For that reason, specific etiologies of 46,XY DSD (401) have a significant risk factor for GCT development (393). Early Sertoli cell development is also disturbed in patients with 45X/46,XY mosaicism (402). The presence of the well-defined Y chromosome region, known as the gonadoblastoma Y locus (GBY), is a prerequisite for malignant transformation. Among the genes located in the GBY region the testis-specific protein Y (TSPY) seems to be the most significant candidate gene for the tumor-promoting process (288,403). The presence of undifferentiated gonadal tissue containing germ cells that abundantly express TSPY has also been identified as a gonadal differentiation pattern bearing a high risk for GCT development (404). Prolonged expression of OCT3/4 (POU5F1) and the stem cell factor KITL after one year of age are also estimated to play a role in GCNIS/GCT development. Other factors implicated in that risk include MAP3K1 variants in 46,XY patients with gonadal dysgenesis due to MAPK signaling pathway upregulation and loss of androgen receptor function in patients with androgen insensitivity syndrome (289,405). Additionally, gonads at the abdominal region are at higher risk of GCNIS/GCT development than those appropriately positioned (393,406).

Unfortunately, GCNIS/GCT screening is challenging due to a lack of a predictive factor or a biomarker with adequate sensitivity and specificity (407). As far as imaging is concerned, ultrasound (US) is more sensitive than MRI at identifying dysgenetic gonads, but MRI showed better sensitivity and specificity than US at localizing non-palpable gonads (408). However, both imaging techniques are poor at identifying GCNIS/GCT, since MRI failed to identify GCNIS in patients with CAIS and the US only identified one out of ten malignant lesions in 46,XY DSD people (409). There are serum markers that are associated with GCT in non-DSD people, such as alpha-fetoprotein, beta-hCG, and lactate dehydrogenase, but there is poor evidence about how useful they are for GCT screening in 46,XY DSD individuals (410). An interesting perspective for GCT screening are microRNAs (miRNA), since some miRNA clusters are expressed in the presence of GCT (411). For non-DSD people, microRNAs are more sensitive than serum markers and imaging to detect GCT. Noteworthy, GCNIS also expresses some embryonic-type miRNAs (miR-371-3, miR-302, and miR-367) that are also expressed by GCTs (410,412). Therefore, they have the potential to serve as a biomarker even for GCNIS(407).

Overall, neoplastic transformation of germ cells in dysgenetic gonads (gonadoblastoma and/or an invasive germ cell tumor) occurs in 20-30% of 46,XY DSD individuals, but the risk varies among 46,XY DSD etiologies (413). Individuals with Denys-Drash syndrome (40%), Frasier syndrome (60%), and gonadal dysgenesis (15 - 35%) have the highest risk of GCNIS/GCT among 46,XY DSD etiologies (413). On the other hand, individuals with CAIS (at prepubertal age) and ovotestis DSD have lower risk of GCNIS/GCT (414). The age matters in the estimation of GCNIS/GCT risk. For example, it is as low as 1.3% in CAIS individuals before puberty, but it can reach 33% thereafter (287,415).

For 46,XY DSD subjects, gonadectomy is classically recommended to avoid GCNIS/GCT development, preventing additional therapies and related risks (290). Despite a very effective strategy to avoid GCNIS/GCT, gonadectomy leads to hypogonadism and infertility.

Regarding the time for gonadectomy, bilateral gonadectomy should be performed in early childhood in 46,XY DSD patients with gonadal dysgenesis, females with Y chromosome material, and patients with androgen biosynthesis defect, unless the gonad is functional and easily accessible to palpation and imaging studies, which should be performed yearly (11,289). Although data are limited, in the androgen insensitivity syndrome the risk seems to be markedly lower in the complete form before puberty than in the other 46,XY DSD (416). Therefore, gonadectomy can be postponed until puberty is complete in CAIS individuals (417). Unfortunately, the GCNIS/GCT risk for other causes of 46,XY DSD patients, such as Leydig Cell Hypoplasia and 5 alpha reductase type 2 deficiency has not been estimated yet.

Rarely, gonadal tumors can produce sexual steroids (418). In those cases that are able to produce estrogens, spontaneous breast development may be a clinical sign that suggests the presence of an estrogen-secreting gonadal tumor, and bilateral gonadectomy is indicated even at early childhood, regardless of the 46,XY DSD etiology.

Overall, 46,XY DSD patients are at increased risk for gonadal malignancy which seems to be related to 46,XY DSD etiology. While it is clear that prepubertal CAIS patients are at low risk for GCT and 46,XY DSD individuals harboring WT1 variants present a high risk for GCT development, the real GCT risk for other 46,XY DSD etiologies is not that clear (413). In the absence of a reliable predictive factor or biomarker of GCNIS/GCT as well as appropriate recommendations for GCT screening, bilateral gonadectomy will still be recommended for most 46,XY DSD etiologies.

FERTILITY IN PATIENTS WITH 46,XY DSD

Most 46,XY DSD individuals face infertility due to abnormal gonadal development, endocrine disturbances, anatomical issues, or prophylactic gonadectomy for malignancy risk (419). However, there has been growing evidence showing that fertility is relevant for several 46, XY DSD people, in addition to the possibility of delaying gonadectomy in some 46,XY DSD etiologies (420). In parallel, fertility preservation technologies have been improved in recent years along with a better social perception of non-traditional family structures (421,422).

In 46,XY DSD, the fertility potential varies depending on the underlying etiology as well as the severity of the condition (421). In this sense, all options for fertility should be discussed considering the 46,XY DSD etiology or the gonadal structure and internal genitalia in those in whom the 46,XY etiology is unknown (420).

For example, individuals with complete gonadal dysgenesis possess uterus, despite lacking gametes (423). Therefore, pregnancy by oocyte donation is an alternative for these patients. On the other hand, male individuals with partial and mild androgen insensitivity often present oligospermia, but biological fertility is possible (334,338).

Overall, there is limited literature about fertility potential among 46,XY DSD people. Successful biological fertility was obtained in a man with PAIS after prolonged high-dose testosterone therapy followed by intracytoplasmic sperm injection (424). The possibility of fertility seems to be more frequent among MAIS since there are six cases of successful fertility (338). There are few reported cases of successful pregnancies and live births in men with 5RD2 deficiency, both spontaneous and with assisted reproductive technology (319,425-427). Biological fertility has also been documented in individuals with nonclassical congenital lipoid adrenal hyperplasia, 3b-HSD2 deficiency, and LHCG receptor defect (420). Conversely, there are no reported cases of biological fertility in individuals with classic congenital lipoid adrenal hyperplasia, cytochrome p450 oxidoreductase deficiency, complete CYP17A1 deficiency, 17b-HSD3 deficiency, and CAIS (419,428).

To estimate fertility potential, a pilot study evaluated the presence of germ cells and the germ cell density in individuals with several 46,XY DSD etiologies (429). In six patients with CAIS, all presented Sertoli Cell nodules and hyperplasia, but germ cells were detected in areas between nodules. All six patients with mixed gonadal dysgenesis and two with ovo-testicular DSD presented germ cells, and ten out of twelve 46,XY DSD patients with unknown etiology presented germ cells in their gonads. On the other hand, germ cells were not found in any of the patients with either complete or partial gonadal dysgenesis. However, the number of germ cells was inversely correlated with age, suggesting that the gonadectomy delay may decrease fertility potential. It needs to be confirmed by more extensive studies, but it indicates that 46,XY DSD fertility potential may be greater than previously thought.

As far as desire for fertility is concerned, a large follow-up study included 1,040 DSD individuals to investigate their fertility preferences (430). The authors reported that 55% of patients expressed a desire to have had fertility treatments in the past or have it in the future, and 40% mentioned that they would like to try new fertility treatment techniques. Additionally, CAIS women reported the possibility of future fertility as one of the reasons to keep their gonads (362).

Indeed, fertility preservation has been primarily assessed in oncology to preserve patients' fertility under gonadotoxic treatments (431). In this sense, cryopreservation of postpubertal testicular tissue is helpful to keep fertility potential in patients having gonadectomy or those before gonadotoxic treatment. As an alternative, cryopreservation of immature testicular tissue containing spermatogonial cells or spermatogonial stem cells can be offered to prepubertal patients (432). These techniques could also be considered for 46,XY DSD patients.

In summary, addressing fertility is essential in 46,XY DSD management. The fertility potential must be discussed considering the 46,XY DSD etiology and the patient’s desire. As assisted fertility and preservation techniques improve, these advancements should be offered and accessible to all 46,XY individuals.

46,XY GENDER IDENTITY DISORDERS

Transgender Women are characterized by the wish to live as members of the female sex with conviction and consistently and progressively efforts to achieve such state. 46,XY gender identity disorders are more frequent among the male sex, although it also occurs in the female sex. Its first manifestations usually start during childhood. If it has a biological basis is still unknown, but some hormonal alterations during intrauterine life and familial factors before and after birth cannot be ruled out (433).

The term used to name men and women who live a relevant incongruence between their gender identity and their inborn physical phenotype has changed over time. The term “trans-sexualism” was coined by Hirschfeld in 1923 and was adopted by the International Classification of Diseases – version 10 (ICD-10). The American Psychiatric Association, in its 4^th edition, adopted “gender identity disorder” to define persons who are not satisfied with their biological gender (Association, American Psychiatric. "Diagnostic and statistical manual of mental disorders (2000).

Finally, the current classification system of the American Psychiatric Association (DSM-5) replaced the term “gender identity disorder” with “gender dysphoria” and the upcoming version of International Classification of Diseases – version 11 (ICD-11) has proposed the term “gender incongruence” (434).

In this chapter we will use the current DSM-5 term, “gender dysphoria”. To refer to male to female gender-dysphoric persons we will use the term transgender woman (American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition - DSM-5; 2013). Therefore, the term “transgender woman” refers to all 46, XY individuals with typical male phenotype who wish to live and be accepted as a female.

Higher prevalence of addictions and suicidal thoughts or suicide attempt than those observed in the general population, revealed the need for early care of these patients by health professionals. Among transgender women, total mortality was 51% higher than in the general population, mainly from increased mortality rates due to suicide, acquired immunodeficiency syndrome, cardiovascular disease, drug abuse, and unknown cause (435). Based on these data, supervised gender-affirming treatment for gender dysphoric persons is crucial because they are at increased risk of committing suicide and self-harm (436).

Management of Adult Transgender Women

As proposed by the Harry Benjamin International Gender Dysphoria Association, now known as World Professional Association for Transgender Health (WPATH), the process of gender-affirming treatment should be given by a multi and interdisciplinary team, in which the endocrinologist has a key role (437).

The interdisciplinary team should consist of a psychologist, a psychiatrist, an endocrinologist, and a surgeon, at least (438). It would be ideal that they all participate in an integrated and consistent way across all the steps of the treatment (439).

The mental health professionals (psychologist and psychiatrist) make a distinction between gender dysphoria and conditions with similar features (body dysmorphic disorder and body identity integrity disorder), decide whether the individuals fulfill ICD-11 and DSM-5 criteria, recommend the appropriate treatment and follow-up before, during and after gender-affirming treatment. The endocrinologist will inform about the possibilities and limitations of all sorts of treatment, initiate and monitor the cross-sex hormonal treatment and participate in the indication of gender-affirming surgery. At the final step, the surgeon performs surgical procedures of the treatment (439).

DIAGNOSTIC ASSESSMENT AND MENTAL HEALTH CARE

Psychological evaluation of persons with gender dysphoria should consider the evolution of the individual as whole, using psychological assessment instruments, such as: freely structured interviews and patterned psychological assessment instruments. For the structured interview, we use a specific questionnaire developed by our mental health professionals that covers childhood, adolescence, and adulthood aspects.

During the psychotherapeutic follow up, besides offering an ideal condition for elaborating conflicts and issues regarding gender identity, other variables should be considered, such as individual general state of mental health, ability and manner of conflict resolution, quality of interpersonal relationships, ability to deal with frustrations and limitations, particularly regarding to surgery’s esthetic and functional results idealization. It is recommended that the relatives and/or spouses are invited for interviews to clear them up upon the offered treatment.

HORMONAL THERAPY FOR ADULT TRANSGENDER WOMEN

Endocrinologists have the responsibility to confirm that persons fulfill criteria for hormonal treatment and clarify the consequences, risks, and benefits of treatment.

Hormone therapy must follow well-defined criteria. The person with gender dysphoria has to: 1) demonstrate knowledge and understanding of the expected and side effects of cross-sex hormone use; 2) complete a real life experience in the gender identity for at least three months, or psychotherapy for a period determined by the mental health professional to consolidate gender identity; and 3) be likely to take hormones appropriately (439).

There are two major goals of hormonal therapy: 1) to replace endogenous sex hormone levels and, thus, induce the appearance of sexual characteristics compatible with female gender identity; 2) to reduce endogenous sex hormone levels and, thereby, the secondary male sexual characteristics and 3) to establish the ideal hormones dosage which allows physiological hormone serum levels compatible with female gender identity by adopting the principles of hormone replacement treatment of hypogonadal patients (439,440).

Hormone therapy provides a strong relief from the suffering caused by the incongruence of the phenotype with the gender identity.

In our clinical practice, we observe that most transgender women consume very high doses of female sex hormones, guided by their wish to obtain fast breast development, and reduce facial hair. However, high doses of hormones are not necessary to achieve the desired effects and are frequently associated with undesirable side effects.

The chosen hormone to induce female secondary sexual characteristics are the estrogens. Several pharmaceutical estrogen preparations, including oral, injectable, transdermal, and intravaginal forms associated or not with progesterone are available. Due to the higher cost of the transdermal preparations, the oral route is the most widely used. Nevertheless, the transdermal route is recommended for transgender women over 40 years of age due to the lower association of transdermal 17β-estradiol replacement with thromboembolic events (441).

Anti-androgens are used as adjuvants to estrogen, especially in the reduction of male sexual characteristics and the suppression of testosterone to levels compatible with the female sex. Cyproterone acetate blocks testosterone binding to its receptor, and in a dose of 50-100 mg/day associated with estrogen can maintain testosterone in female levels in transgender women (442).

At the time, most of the patients in our clinic used conjugated equine estrogens at a dose of 0.625-1.25 mg/day associated with 50 mg/day of cyproterone acetate for an average period of 11 years. At clinical examination we observed satisfactory breast development, decrease of spontaneous erections, thinning of facial and body hair (especially after association with cyproterone acetate), body fat redistribution, enlargement of the areola and nipple and reduction of testicular volume (440).

Testosterone levels remained at pre- or intra-pubertal female range (< 14-99 ng/dL) in all patients; LH levels were pre-pubertal (<0.6-0.7 U/L) in 72% of the cases, and the FSH levels were suppressed (<1.0 U/L) in 40% of cases. Therefore, daily use of oral conjugated estrogens at low doses in association with cyproterone acetate is effective in suppressing the hypothalamic-pituitary-testicular axis in transgender women (440).

Venous thromboembolism may be a serious complication related to estrogen therapy, particularly during the first year of treatment, when the incidence of this event is 2-6% falling to 0.4% in the second year, significantly higher when compared to the overall young population (0.005 to 0.01%/year). This high incidence of thromboembolic events in transgender women seems to be more associated with ethinyl estradiol than natural oral or transdermal estrogens (441). All patients on estrogen therapy have a mild prolactin level increase. However, a small percentage of these subjects have galactorrhea. In our cohort, two patients had macroprolactinoma, which totally regressed with dopamine agonist treatment. Both had previously used high doses of estrogen (443). Endocrinologists should monitor weight, blood pressure, breast enlargement, body hair involution, body fat redistribution and testicular size every six months. The laboratory evaluation should include measurement of LH, FSH, testosterone, estradiol, prolactin, liver enzymes, complete blood count, coagulation factors, and lipid profile. Bone densitometry and breast ultrasound should be performed yearly.

After surgery in patients over 50 years old, the measurement of PSA should be conducted yearly (440).

The current key issues include avoiding supraphysiological doses of estrogen and the use of ethinyl estradiol. The preference should be given to conjugated estrogens or transdermal natural estrogen, especially in patients over 40 years of age (444). Hormone therapy provides a strong relief from the suffering caused by gender dysphoria. (440).

MANAGEMENT OF PATIENTS WITH 46,XY DSD

It is important to stress that the treatment of 46,XY DSD patients requires an appropriately trained multi-disciplinary team. Early diagnosis is important for better outcomes and should start with a careful examination of the newborn’s genitalia at birth (445-447).

Psychological Evaluation

It is of crucial importance to treat DSD patients (448). Every couple that has a child with atypical genitalia must be assessed and receive counseling by an experienced psychologist, specialized in gender identity, who must be act as soon as the diagnosis is suspected, and then follow the family periodically, more frequently during the periods before and after genitoplasty (449,450).

Parents must be well informed by the physician and psychologist about sexual development (451). A simple, detailed, and comprehensive explanation about what to expect regarding integration in social life, sexual activity, need of hormonal replacement and surgical treatment and fertility issues should also be discussed with the parents, before sex assignment (11).

The sex assignment must consider the etiological diagnosis, external genitalia, cultural and social aspects, sexual identity and the acceptance of the assigned gender by the parents (452). In case parents and health care providers disagree over the sex of rearing, the parents’ choice must be respected. The affected child and his/her family must be followed throughout life to ascertain the patient’s adjustment to his/her social sex.

Hormonal Therapy

Sex steroid replacement is an important component of management for some types of 46,XY DSD (453,454). The goals of replacement include induction and maintenance of secondary sex characteristics as well as other aspects of pubertal development including growth. Bone mineral optimization and promotion of uterine development may also be helped by treatment with sex steroids for some patients. Hormone replacement can also impact psychosocial and psychosexual development, as well as general wellbeing, in positive ways for some people (455,456). Induction and maintenance of pubertal development is necessary in most patients affected by 46,XY DSD regardless of male or female rearing; however, specific indications depend on the underlying etiology of the condition.

The purpose of the hormonal therapy is the development of female sexual characteristics and menses in the patients with uterus. There are several options available for estrogen replacement as well as different combinations and doses of progestins (457) however, 17β-estradiol (oral or transdermal) is preferred. Estrogen therapy should be initiated at a low dose (1/6 to 1/4 of the adult dose) to avoid excessive bone maturation in short children and increase gradually at intervals of 6 months. Doses can then be adjusted to the response (Tanner stage, bone age), with the aim of completing feminization gradually over a period of 2–3 years. In 46,XY females with tall stature, adult estrogen dosage is recommended to avoid high final stature. Transdermal delivery avoids hepatic first-pass metabolism resulting in less thrombogenicity and more neutral effects on lipids (458,459). It is also easier to administer small doses of estrogen by cutting up a patch or by using a metered-dose gel dispenser. An initial recommended dose of oral 17-βestradiol is 5 μg/kg daily, titrated every 6–12 months to an additional 5 μg/kg daily until an adult dose of 1–2 mg daily is achieved (459) .

In case of transdermal replacement, the initial recommended dose for the 17-β estradiol patch is 3.1–6.2 mg/24h overnight (1/8–1/4 of 25 mg/24h patch). Transdermal doses can increase 3.1–6.2 mg/24h every 6 months until an adult dose of 50-100 mg/24 h twice a week is achieved (460) Once breast development is complete, an adult dose can be maintained continuously (11). For patients who do not have a uterus, estrogen alone is indicated (458,461). Progesterone is needed to induce endometrial cycling and menses in patients with a uterus. For the latter group, medroxyprogesterone acetate (5 to 10 mg/day) or micronized progesterone (200 mg/day from the 1^st to the 12^th day of each month) are appropriate to maintain uterine health.

Some females with CAIS report decreased psychological wellbeing and sexual dissatisfaction following bilateral gonadectomy and subsequent estrogen replacement (462,463).

Testosterone treatment has been proposed as an alternative to estrogen for hormone replacement in these women and such treatment improves sexual desire (464). However, long-term follow-up studies on the impact of T replacement on additional psychological measures, as well as on bone metabolism and cardiovascular outcomes, are needed (465).

The dilation of the blind vaginal pouch with acrylic molds (325) or exceptionally surgical neovagina promote development of a vagina adequate for sexual intercourse after 6-10 months of treatment when patients desire to initiate sexual activity (466).

For those raised male, T replacement should strive to mimic masculine pubertal induction between 10 and 12 years of age, provided the child’s predicted height and growth are normal and he indicates a desire and readiness for puberty (5). Intramuscular, short-acting injections of T esters are the most suitable formulation to induce male puberty, although other options include oral T undecanoate and transdermal preparations (467,468).. The initial dose of short-acting T esters is 25–50 mg/month intramuscularly, with further increments of 50- 100 mg every 6–12 months, thereafter. After reaching a replacement dose of 100–150 mg/month, the delivery interval can decrease to every two weeks.

An adult dose of 200-250 mg every two weeks (short-acting T esters), 1000 mg every 10-14 weeks (long-acting T esters), or 50-100 mg for T gel or other transdermal preparations applied topically are effective to maintain male secondary sex characteristics (12,468). Monitoring of T levels should be performed on the day preceding the next hormone administration, and serum levels should fall just above the lower limit of the normal range for eugonadal men.

In male patients with androgen insensitivity, higher doses of testosterone esters (250-500 mg twice a week) are used to increase penile length and male secondary characteristics. Maximum penis enlargement is obtained after 6 months of high doses and after that, the normal dosage is re-instituted (272,313). The use of topical DHT gel is also useful to increase penile length with the advantage of not causing gynecomastia.

Glucocorticoid Replacement

It is necessary for 46,XY DSD patients with classical forms of congenital lipoid adrenal hyperplasia, POR, 3β-HSD type II deficiency to receive glucocorticoid replacement for adrenal insufficiency and in 17α-hydroxylase/17,20-lyase deficiency for hypertension management (469) (267).

Mineralocorticoid replacement is also required for 46,XY DSD salt-losing patients (470).

Surgical Treatment

Surgical approach for 46,XY DSD patients includes: gonadal management, removal of internal structures that disagree with the social sex and reconstruction of the atypical external genitalia. Genital reconstruction involves the feminization or masculinization of external genitalia; these procedures are being widely discussed and controversy continues over the ideal age for genital surgery (471,472). There is a lack of data concerning this issue: a survey with 459 individuals (≥ 16 years) with a DSD diagnosis concerning patients desire about timing of genital surgery was published (473). A total of 66% of individuals with CAH and 60% of those with 46,XY DSD thought that infancy or childhood were the appropriate age for genital surgery. This report concluded that case-by-case decision-making is the best approach (473). In our experience, patients submitted to surgery in adulthood, preferred surgery in infancy and none of the patients operated during childhood regretted the surgery at that age (474).

Laparoscopy is the ideal method of surgical treatment of the internal genital organs in patients with 46,XY DSD (475). In these patients, the indications for laparoscopy are the removal of gonads and ductal structures that are contrary to the assigned gender and the removal of dysgenetic gonads, which are nonfunctional and present potential for malignancy. In addition to being a minimally invasive surgery, one of the main advantages of this method is the lack of scars.

Feminizing genitoplasty includes the reduction of enlarged clitoral size, opening the urogenital sinus to separate the urethra from the vaginal introitus, and constructing labioscrotal folds. Feminizing techniques have evolved over time to achieve better cosmetic outcomes (476,477). Many techniques have been proposed to separate the urethra from the vaginal introitus and bring both to the surface of the perineum. Fortunoff and Latimer in 1964 described the most commonly used technique until the present day, using an inverted U-shaped perineal skin flap to enlarge the vaginal introitus allowing adequate menstrual flow and future sexual activity (478). Failure to interposing an adequate flap will result in persistent urogenital sinus or vaginal introitus stenosis, requiring later revision (479). Vaginal dilation with acrylic molds in patients with short vagina or introitus stenosis showed to be a good treatment choice when these patients wished to start sexual intercourse, resulting in better outcomes (325). To reduce clitoral enlargement a number of techniques were proposed during the years (480). Kogan described preserving the neuro-vascular bundle attached to the dorsal portion of the tunica albuginea to protect the nerves and blood supply (481) and this is the technique of choice. The redundant clitoral skin obtained during clitoroplasty is used to create the labia minora; this skin is divided longitudinally and then sutured along either side of the vagina. When necessary, the reduction of labioscrotal folds is performed to create the labia majora, often using a Y-V plasty technique (482).

The most common surgical complications, in feminizing genitoplasty, includes: clitoral ischemia or necrosis that can rarely occur in patients with high grade of virilization; introitus stenosis or vaginal stenosis particularly when the confluence of the vagina and urethra is far from the perineum surface and urinary infections mostly observed in patients with persistence of urogenital sinus (471,479,480).

In order to minimize surgical complications and dissatisfaction in adulthood, only skilled surgeons with specific training should perform these procedures in the DSD patients (8). In our experience, the single-stage feminizing genitoplasty consisting of clitoroplasty with the preservation of dorsal nerves and vessels and ventral mucosa, vulvoplasty and Y-V perineal flap, followed by vaginal dilation with acrylic molds, allowed good cosmetic and functional results (483).

For the males, masculinizing procedures aims to allow the patient to have micturition standing up without effort with a straight and wide stream and to have a satisfactory sexual life with straight erections. The genital surgery consists in correction of hypospadias and scrotal abnormalities, relocation of the testes to the scrotum or removal when dysgenetic, and resection of Mullerian remnants (326,484). Correction of hypospadias includes correction of phallic curvature (orthophalloplasty) and construction of a urethra to the tip of the glans (urethroplasty). Preoperative administration of testosterone is indicated for patients with a small penis (485). Usually, multistage procedures are preferred for male genital reconstruction in DSD patients, due to the severe under virilization represented by proximal hypospadias with severe curvature. The first stage repair consists in ortho-phaloplasty and scrotoplasty (486) (487). The second stage is performed 6-9 months later and consists in urethroplasty). The two-stage approach typically results in better cosmetic outcomes and fewer postoperative complications for patients with severe hypospadias and significant chordee (326,487-489). The most frequent complication in correction of hypospadias is urethral fistula (23%) followed by urethral strictures (9%) and diverticula (4%) (490). This frequency is highly variable in the literature (490). Fistula can be observed just after surgery or months later, but urethral stenosis in some cases can occur several years after surgery. Reoperations are necessary to correct fistula, diverticula and particularly to treat severe urethral strictures. The buccal mucosa graft is commonly used to enlarge the urethra in these cases (490). For patients with undescended testes, simultaneous orchidopexy may be performed. The surgical treatment of gonads of 46, XY DSD patients aims to preserve testicular function (production of testosterone and sperm) and prevent malignancy (288,360,491). Finally, gonadectomy is recommended for patients at risk for neoplastic transformation of germ cells (gonadoblastomas and/or an invasive germ cell tumor) in dysgenetic gonads (287).

When gonadectomy is recommended, patients may then choose to have a testicular prosthesis placed in the scrotum (492).

Müllerian structures are rudimentary in some patients and present as a cystic prostatic utricle. These utricles may be left in situ when asymptomatic, but in cases of recurrent urinary tract infection, stones, or significant post-void urethral dribbling due to urinary pooling, they can be removed either laparoscopically or through a sagittal posterior incision of the perineum (475). With either approach, great care must be taken to prevent injury to the vas deferens, seminal vesicles and pelvic nerves so as to avoid subsequent infertility, erectile dysfunction and urinary incontinence (488,493). Late evaluation of 46,XY DSD patients operated in childhood due to proximal hypospadias reveals that many felt that their genitals had an unusual appearance or presented some degree of urinary or sexual dysfunction (494). Objectively, most DSD patients have a penile length below the -2.0 SD (5.2 ± 2.0 cm) (326). Dysfunctional voiding and lower urinary tract symptoms are also more frequent in these patients than in controls (495). However, between 55.6 and 91% of these patients after genitoplasty were satisfied with their overall sexual function after genitoplasty, when considering sexual contacts, libido, erections, orgasm, as well as size of the penis and volume of ejaculation (326),(494),(496),(497),(498). The long-term outcomes were evaluated for a long time concerning functional and cosmetic results that could be analyzed by objective criteria. The subjective long-term evaluation analyzing psychological and sexual implications in quality of life were often neglected in the past, but is being currently explored (332) (499) (500). Jones et al reported that 81% were satisfied with their genital appearance and that 90% were satisfied with their overall body image (500). Most of our patients were satisfied with their genital appearance and present satisfactory sexual performance as long as they present a penis size of at least 6 cm (326).

ACKNOWLEDGMENT

The authors would like to thank the postgraduate students Nathalia Lisboa Gomes and Jose Antonio D Faria Junior for their help in the update of this chapter.

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Pituitary and Hypothalamic Tumor Syndromes in Childhood

ABSTRACT

Central nervous system (CNS) tumors are the second commonest childhood malignancy, with 10% of these affecting the suprasellar and/or intrasellar regions. Survival has increased significantly over the last decade as a result of improved multimodality cancer therapies and better supportive care. Measurements of serum prolactin, α-fetoprotein, and β-hCG as well as baseline pituitary function tests are essential at diagnosis prior to commencement of any therapy. Craniopharyngiomas and low-grade gliomas account for most of these tumors, whilst other histological subtypes such as pituitary adenomas, germinomas, and hamartomas are rare. Non-neoplastic masses include pituitary hyperplasia and Rathke’s cleft cysts. Neurological syndromes and endocrine dysfunction are often present at diagnosis, and may be missed if not sought for. Post-diagnosis, endocrinopathies can evolve over decades secondary to tumor and/or treatment, necessitating long-term follow-up of such patients. Treatment of endocrine dysfunction is crucial not just to avoid the fatal consequences of untreated secondary adrenal insufficiency and/or diabetes insipidus, but also to improve quality of survival, and should be closely supervised by a pediatric endocrinologist with experience in the management of such patients. Growth hormone therapy in replacement doses in particular has not been shown to increase the risk of tumor recurrence. The “hypothalamic syndrome”, including variable hypothalamic dysfunction (e.g., sleep-wake cycle disturbances, temperature dysregulation, adipsia, and behavioral disorders) and hypothalamic obesity, is a common and as yet untreatable sequela of both tumor and treatment. The latter is caused by dysregulation of a network anorexigenic and orexigenic hormone signals which is only beginning to be elucidated.

INTRODUCTION

Central nervous system (CNS) tumors are the second commonest childhood malignancy after leukemias, accounting for 25% of cancers in children <15 years of age with an annual incidence rate of 35 cases/million/year (1–4). As with all childhood cancers, their incidence is gradually increasing worldwide (1,2,5), an effect largely attributed to improvements in diagnosis and tumor registration (6–8), and more recently campaigns such as the UK HeadSmart project aimed at increasing awareness of pediatric brain tumor symptoms (http://www.headsmart.org.uk/) (9). Concurrently, 5-year survival for CNS tumors has increased much more steeply from 57% to 65% in the last decade (~95% in low-grade gliomas) as a result of improved multimodality cancer therapies and better supportive care (10–12).

However, while survival is high, increasingly intensive treatment strategies aimed at improving cure in a small minority can conversely cause a higher toxicity burden in the larger majority, with a rapidly accruing cohort of survivors faced with reduced quality of life due to late and evolving multi-organ toxicities (13–15). Over 40% of these chronic morbidities (“late effects”) are severe, disabling or life-threatening (16), and more than 80% of CNS tumor survivors develop at least one endocrinopathy, most frequently growth hormone deficiency (17). Indeed, suprasellar tumors have been found to be the commonest cause of hypothalamo-pituitary dysfunction in adult cohort studies (18,19). However, when compared with adult CNS tumors, pediatric tumors tend to be more curable, and the early presentation of some tumors (e.g., craniopharyngiomas, primitive neuroectodermal tumors (PNET)), and their association with mutations in neural development genes blur the delineation between congenital malformations and neoplasia (20–22).

Tumor location and histology is distinctly age-dependent: 30% of tumors under the age of 14 years are infratentorial (medulloblastomas, posterior fossa juvenile pilocytic astrocytomas, and ependymomas), whilst 26% and 16% of tumors diagnosed in young adulthood (15 to 24 years) are supratentorial or suprasellar respectively (non-pilocytic astrocytomas, other gliomas, pituitary adenomas, and germinomas) (4,23). Supra- and intrasellar tumors constitute 10% of all pediatric CNS tumors (23,24) and their close proximity to the vital hypothalamo-pituitary axis (HPA) increases the risk of important endocrine dysfunction. This may occur secondary to tumor mass effect and/or treatment, and can therefore be manifest at presentation or evolve subsequently during or after completion of oncological therapies. Dissecting the effect of tumor from treatment on endocrinopathies diagnosed after commencement of therapy is particularly complicated. We aim here to (1) outline the epidemiology, clinical features, and management of common pediatric suprasellar tumors not readily addressed in other chapters, (2) examine the common clinical neuroendocrine presenting features and (3) summarize common themes in the neuroendocrine late effects observed at follow-up of these patients.

THE DIFFERENTIAL DIAGNOSIS OF PEDIATRIC SUPRA- AND INTRASELLAR MASSES

The definitive diagnosis of pediatric suprasellar and intrasellar masses is crucial, as therapeutic strategies differ markedly depending on histological subtype. However, a tissue diagnosis may not always be possible due to their location, as even minor procedures such as biopsies can lead to life-threatening endocrinopathies such as diabetes insipidus (DI) (25). Biochemical measurements of serum prolactin (PRL), α-fetoprotein (AFP), and β-human chorionic gonadotrophin (β-hCG) to aid the diagnosis of prolactinomas and secreting germinomas respectively are therefore absolutely essential prior to commencement of any therapy.

Table 1. The Differential Diagnosis of Pediatric Suprasellar Tumors and Other Disorders

Neoplastic

Craniopharyngioma

Low-grade glioma (mainly pilocytic astrocytoma)

Pituitary adenoma

Germ cell tumor (mainly germinoma)

Hamartoma

Meningeal metastases

Non-neoplastic

Pituitary hyperplasia

Pituitary stalk thickening

Langerhans cell histiocytosis*

Tuberculosis

Sarcoidosis

Rathke cleft cyst

Arachnoid cyst

Epidermoid/dermoid cyst

Meningioma

*The classification of Langerhans cell histiocytosis as a non-neoplastic disease is debatable.

Craniopharyngiomas

Figure 1. T₁-weighted MRI images of a craniopharyngioma demonstrating the coexistence of solid, cystic and calcified components with the tendency for multiple progressions over seven years. (a) After initial endoscopic cyst fenestration and ventriculoperitoneal shunt insertion, (b) after first transcranial debulking, (c) first cystic progression, (d) after first cyst drainage via reservoir, (e) second cystic progression, (f) after second transcranial debulking, (g) after adjuvant radiotherapy and third cystic progression, (h) after second cyst drainage via reservoir, (a) after fourth cystic & solid progression, (j) after complete resection.

Craniopharyngiomas are by far the commonest suprasellar tumor of childhood, accounting for up to 50-80% of masses in this region (24,26–28) and 1.5-11.6% of all pediatric CNS tumors (3,24,26,29,30). There is a bimodal age distribution in incidence, with the peak incidence in childhood occurring between the ages of 5-14 years at 1.4 cases/million/year (29,31). They are benign tumors originating from the embryonal epithelium lining Rathke’s pouch and are almost invariably adamantinomatous in childhood, characterized by the presence of intratumoral calcifications(32). Over-activation of the Sonic hedgehog (SHH) and Wnt/β-catenin pathways, both important in both pituitary stem cell development and carcinogenesis, have been shown to be key to their formation (20,21), but they occur typically sporadically, with only one case report of familial adamantinomatous craniopharyngiomas occurring in a consanguineous pedigree reported in the English literature (33). Contrastingly, papillary craniopharyngiomas are found almost exclusively in adults and harbor the BRAF V600E mutation instead (34).

Symptoms related to hypothalamo-pituitary dysfunction, such as weight gain, growth failure, prolonged recovery from infections, and abnormalities of puberty are often under-recognized but in fact constitute the third commonest group of clinical findings at diagnosis, after symptoms related to raised intracranial pressure (e.g., headaches, vomiting) and visual deterioration (22,35–47). Radiologically, 65-93% of these tumors are calcified but a plain X-ray or computerized tomography (CT) scan may be required to demonstrate this. The coexistence of solid, cystic, and calcified structures on neuroimaging, as well as the characteristic cholesterol crystals seen under microscopy of the “engine fluid” aspirated surgically from cystic components are so highly suggestive of the diagnosis that histological confirmation from biopsies of solid components may be unnecessary, particularly as this may further compromise hypothalamo-pituitary function (32,48). Anatomically, 75% of craniopharyngiomas are suprasellar with an intrasellar extension, 20% are exclusively suprasellar, and 5% are exclusively intrasellar, with over 50% involving the hypothalamus and nearly one-third invading the floor of the third ventricle (26,37,44).

Due to their location, a significant proportion of these tumors are not completely resectable, but their relative rarity, high rates of survival, and benign histology have precluded them from pan-European randomized trials, resulting in a lack of agreement on the optimal treatment strategy. Most recently, the first evidence- and consensus-based national UK guideline for the management of craniopharyngiomas in children and young people has been published by the UK Children’s Cancer and Leukemia Group (CCLG), with endorsement from the Royal College of Pediatrics and Child Health (RCPCH) and British Society of Pediatric Endocrinology & Diabetes (BSPED) (49).Importantly, these guidelines advocate a more conservative approach to the degree of surgical resection in the presence of significant hypothalamic involvement in order to minimize further damage to the hypothalamo-pituitary axis (39,50,51), balanced against the need to relieve symptoms of raised intracranial pressure, preserve vision, and provide long-term control and reduced recurrence rates (49,52,53). The use of adjuvant radiotherapy in combination with subtotal tumor resection has been shown to achieve survival rates which are on par with complete tumor resection (5-year progression-free survival 73-100% vs 73-82%), with the potential for less neuroendocrine dysfunction (54–56). More recently, the use of proton beam therapy has increased, with equivalent survival outcomes to conventional radiotherapy, but there remains the issue of insufficient follow-up data to ascertain its long-term toxicity profile (57,58). Experience with systemic or intracystic chemotherapy, intracystic interferon, and radioisotope instillation of ³²P or ⁹⁰Y have been met with conflicting success and cannot therefore be currently recommended as primary treatment approaches in children (59–62). Ultimately, despite high long-term overall survival (80% at 30 years), (37) up to 98% of survivors experience dysfunction in at least one hypothalamo-pituitary axis with high rates of morbid obesity(45,63).

Low-grade Gliomas (LGGs)

Figure 2. T₁-weighted MRI image demonstrating appearances of a large, lobulated optic pathway astrocytoma with hydrocephalus and widespread leptomeningeal dissemination affecting the brainstem, cerebellum, and spinal cord.

LGGs account for >40% of all CNS tumors and are thus the commonest pediatric intracranial tumor (3,8). The optic pathway, hypothalamus, and suprasellar midline are the second most frequent location for LGGs (30-50%) after the cerebellum, cerebral hemispheres, and brainstem (12,64). Even in the suprasellar region they are the second commonest pediatric tumor after craniopharyngiomas, and are similarly regarded as benign (grade I or II), the vast majority being juvenile pilocytic astrocytomas (65). The genetic tumor predisposition syndrome neurofibromatosis type 1 (NF-1) is present in 10-16% of cases, whilst 15% of asymptomatic NF-1 children will have LGGs on neuroimaging. NF-1-associated tumors more often originate from the optic nerves (70%) than from the hypothalamochiasmatic area (27-40%) and tend to a more indolent course (11,12,64,66–69). Mutations involving KIAA1549, BRAF and Ras proto-oncogenes are associated with pilocytic astrocytomas and disruptors targeted at these pathways form the basis of current clinical therapeutic trials (70–72). Similar to craniopharyngiomas, the commonest symptoms at diagnosis are related to visual changes or raised intracranial pressure, with disorders of the LH/ FSH axis being the most prevalent endocrinopathy at presentation (25,66,73–75). In infancy, hypothalamic LGGs can present with diencephalic syndrome (see below) (11,76–78), which significantly increases the risk of future neuroendocrine dysfunction (79).

Complete tumor resection has been shown to be a favorable risk factor for survival (12,64) but suprasellar and/or optic pathway tumors cannot be completely resected without causing major visual and neuroendocrine morbidity. Treatment trials have thus focused on medical strategies, with radiotherapy being delayed in favor of chemotherapy in young children due to concerns of cognitive dysfunction (80), subsequent primary cancers (SPCs) (81,82) and radiation-induced vasculopathies (83), despite showing superior 5-year progression-free survival rates (65% vs. 47%) (11). However, to date none of the previous international treatment trials – LGG1 (1997-2004) or LGG2 (2005-2010) – were randomized, these being purely observational studies aimed at improving visual outcomes but with little reported success (11,12,84). At the time of writing, the first randomized interventional study of chemotherapeutic strategies (LGG3) is being designed with careful long-term prospective measurements of visual and neuroendocrine outcomes. More recently, tumors harboring BRAF mutations have been the target of MAPK/ERK kinase (MEK) and BRAF inhibitors such as trametinib and dabrafenib (72,85–87), although these can still lead to various side effects including endocrinopathies(88).

A 30-year survival analysis has revealed the extent of long-term neuroendocrine dysfunction affecting these patients with new endocrine deficits appearing up to 15 years post-diagnosis, and 20-year endocrine event-free survival approaching 20% (25). Hypothalamic tumor location is a more important independent risk factor for long-term anterior hypothalamo-pituitary deficits than radiotherapy exposure; however only surgical intervention (regardless of extent) has been shown to be independently associated with posterior pituitary dysfunction and life-threatening salt and water imbalances (25,64). Similar to craniopharyngiomas, overall survival is high (85% at 25 years), but ~80% of survivors experience at least one endocrinopathy (25,79).

Pituitary Adenomas

Figure 3. T₁-weighted MRI image demonstrating appearances of a giant prolactinoma. There is obscuration of normal pituitary morphology due to the tumor arising from the pituitary gland itself.

Pituitary adenomas are rare in childhood, accounting for just 3% of all supratentorial tumors with an estimated annual incidence of 0.1 cases/million/year in children (89). The vast majority are functioning, with prolactinomas alone accounting for 50% of adenomas and 2% of all pediatric and adolescent intracranial tumors. Therefore, the measurement of plasma prolactin (PRL) may be diagnostic and is absolutely mandatory prior to planning surgery for any pituitary mass, as medical treatment alone may be entirely curative (90,91). ACTH- and GH-secreting adenomas are the next commonest, whilst TSH-secreting, gonadotrophin-secreting, and non-functioning adenomas are vanishingly rare (91–93).

A child with a pituitary adenoma may be the index case for a genetic tumor predisposition syndrome (up to 22%), particularly given their rarity, and therefore careful documentation of their family history and testing for multiple endocrine neoplasia type 1 (MEN1) and aryl-hydrocarbon receptor interacting protein (AIP) gene mutations are therefore paramount in all cases (94–96). Other genetic syndromes associated with pituitary adenomas that need to be considered are multiple endocrine neoplasia type 4 (CDKN1B), Carney complex (PRKAR1A), McCune-Albright syndrome (GNAS), SDH-related pituitary adenoma syndrome (SDHB, SDHC, SDHD), and DICER1 syndrome (97).

Investigation and management of pituitary adenomas depends on whether they are functioning or non-functioning, and in the case of the former, which hormones are being secreted in excess. Similar to craniopharyngiomas, an evidence- and consensus-based national UK guideline is being written for the management of pituitary adenomas in children and young people as a collaborative effort between the CCLG, RCPCH and BSPED.

PROLACTINOMA

Pituitary adenomas are classified as microadenomas (<1 cm), macroadenomas (>1 cm), and giant adenomas (>4 cm). In prolactinomas plasma PRL levels generally, but not exclusively, increase with tumor size. Hyperprolactinemia may also result from stalk compression by tumor mass (interrupting hypothalamic dopaminergic inhibition of PRL secretion) and antipsychotic medication but PRL concentrations are usually <2000 mU/l and patients rarely symptomatic (98). Laboratories should always screen for artefactual hyperprolactinemia due to macroprolactin, but levels >5000 mU/l are usually diagnostic and symptomatic. Occasionally, falsely low results can be due to interference by extreme hyperprolactinemia on antibody-antigen sandwich complex formation, a phenomenon known as the hook effect. In cases of large tumors, samples should therefore be diluted 100-fold and repeated for confirmation (99). Clinical presentation varies according to the size of tumor, gender, and pubertal status, with girls usually experiencing galactorrhea, pubertal delay, or amenorrhea and boys presenting later with larger, more aggressive tumors with raised intracranial pressure (90).

Given the paucity of good quality outcome data in children, treatment guidelines follow those for adults (53,91), recommending dopamine agonists (DAs) as first line, ideally cabergoline due to its high efficacy and tolerability (98). Starting doses, dose escalation and duration of therapy in children remain undefined and are critical questions given the potential for more aggressive disease and cardiac valve abnormalities with long-term cumulative exposure (100). Surgery should be reserved for those cases resistant to DAs or for neurosurgical emergencies (e.g., neuro-ophthalmic deficits, pituitary apoplexy) and both trans-sphenoidal and transcranial approaches should be considered by an experienced pediatric neurosurgeon. Radiotherapy has usually been reserved for treatment failures in view of the presumed risk of post-treatment endocrine morbidity and second primary cancers. However, the former may have been overestimated in view of the high incidence of endocrinopathies already present at diagnosis (101), and therefore this treatment modality should be considered earlier and prior to other more experimental treatments such as temozolomide chemotherapy (98). As with other hypothalamo-pituitary tumors, long-term neuroendocrine and secondary cardiovascular morbidity is significant (102).

CORTICOTROPHINOMAS

The age distribution for corticotrophinomas is younger than that of prolactinomas (where the incidence increases in adolescence and young adulthood), with Cushing disease accounting for the vast proportion of Cushing syndrome in children aged >5 years, and >70% of pituitary adenomas in the prepubertal age group (103,104). These tumors are nearly always microadenomas. Common presenting features include weight gain with linear growth arrest or short stature, change in facial appearance, fatigue, striae, hirsutism, emotional lability, hypertension, acne, headaches, or psychosis (104–106). Diagnosis is achieved by firstly screening for Cushing syndrome indicated by a raised urine free cortisol (sensitivity 89%) or midnight cortisol concentration of >50 nmol/l (sensitivity 99%, specificity 20%). This is then followed by a low-dose (sensitivity 100%, specificity 80%) then high-dose (sensitivity 94%, specificity 70%) dexamethasone suppression test (104,107–111). CRH-stimulated bilateral inferior petrosal sinus sampling (BIPSS) may help successfully localize the position of the microadenoma (104,105). Transsphenoidal resection is the first-line treatment of choice, superseding bilateral adrenalectomy which carries a risk of post-operative Nelson syndrome(112). Cure rates are 45-78% with nearly 40% requiring adjuvant radiotherapy (113–115).

SOMATOTROPHINOMAS

8-15% of all pituitary adenomas in patients <20 years of age secrete GH, with a significant proportion co-secreting PRL and TSH (103,116). Genetic syndromes associated with somatotrophinomas include MEN-1 (MEN1), Carney complex (PRKAR1A), McCune-Albright syndrome (GNAS), and familial isolated pituitary adenoma (FIPA, AIP) syndrome (97). Due to the absence of complete epiphyseal fusion, in childhood and adolescence, somatotrophinomas present with pituitary gigantism rather than acromegaly. Tall stature and increased growth velocity however can still be associated with other acromegalic features such as mild obesity, macrocephaly, acral enlargement, frontal bossing, and macrognathia (93,117). Investigations reveal high random GH and IGF-1 concentrations, loss of GH pulsatility, and failure of GH suppression to an oral glucose tolerance test (87). Like corticotrophinomas, transsphendoidal resection is the treatment of choice but a significant proportion of patients require adjuvant medical therapy with somatostatin analogues (octreotide, lanreotide), dopamine agonists (cabergoline, bromocriptine), or the GH receptor antagonist pegvisomant (118). Radiotherapy has been used with limited effect (119).

Germ Cell Tumors

Figure 4. T₁-weighted MRI image demonstrating the appearance of a contrast-enhancing suprasellar β-hCG-secreting germinoma in a patient who presented with central diabetes insipidus.

Germ cell tumors (GCTs) are tumors arising from primordial germ cells normally sited in the testes and ovaries and can be subclassified into germinomatous (GGCT, usually non-secreting but can occasionally produce βhCG) and non-germinomatous germ cell tumors (NGGCT). NGGCTs and can be further classified into yolk sac tumors (secreting α-fetoprotein (AFP)), choriocarcinomas (secreting βhCG), and embryonal carcinomas. In contrast to craniopharyngiomas and LGGs, intracranial GCTs account for just 3-4% of all primary pediatric and young adult CNS tumors <24 years (23,120). There is a clear peak in incidence in adolescence and young adulthood, with age-adjusted incidence rates rising from 0.9 cases/million/year in patients <10 years to 1.3-2.1 cases/million/year in patients aged 15-24 years (23,120). Boys are affected nearly three times as often as girls, and this sex distribution is magnified in adolescence (male: female ratio of >8:1) (23). GCTs are also the commonest CNS tumor in Klinefelter and Down syndromes (121). Diabetes Insipidus (DI) and gonadotrophin-independent precocious puberty (due to βhCG acting on the LH receptor) are common findings at diagnosis and present in 30-50% and 11-12% of patients respectively. Unlike NGGCTs, GGCTs can grow indolently (if at all), meaning that both clinical and radiological features can often be subtle at onset, and delays in diagnosis up to 21 years have been reported (122–124).

Histologically, intracranial GCTs resemble their gonadal counterparts (ovarian teratoma or testicular seminoma) and account for 34% of all such tumors (125). They have a particular predilection for the pineal gland (37-66%) and suprasellar region (23-35%), such that synchronous (bifocal) pineal and suprasellar tumors are pathognomonic. Both GGCTs and NGGCTs are extremely chemo- and radiosensitive, and their propensity to metastasize throughout the cerebrospinal fluid (26,121,126) has meant that whole neuraxial (craniospinal) irradiation has been standard therapy for decades, with overall and progression-free survival rates approaching 100% (119). Chemotherapy alone has been shown to result in inferior survival (127), and more recent attempts to reduce the irradiation field with adjuvant chemotherapy in an effort to preserve cognitive function have shown little reduction in overall survival (121,128,129). The latest SIOP CNS GCTII however aims to reduce the radiation dose and field by stratifying treatment strategies between NGGCT and GGCTs, and based on the absence or presence of metastatic disease (https://www.skion.nl/workspace/uploads/2_siop_cns_gct_ii_final_version_2_15062011_unterschrift_hoppenheit.pdf). As for other suprasellar tumors, the rate of post-treatment endocrine morbidity is significant, with 50-60% of patients having at least one endocrinopathy (122).

Hypothalamic Hamartomas

Figure 5. T₁-weighted MRI image demonstrating the appearances of a pedunculated hypothalamic hamartoma (arrowheads) arising from the floor of the third ventricle in a patient who presented with central precocious puberty. The pituitary morphology is otherwise normal.

Hypothalamic hamartomas are extremely rare congenital (rather than neoplastic) malformations consisting of grey matter heterotopia in the tuber cinereum and inferior hypothalamus (24,26,130). Their true prevalence is unknown but is estimated to occur in between 1 in 50,000 – 1 million individuals (131–133). Symptom onset occurs in infancy to early childhood, with the mean age of first seizures occurring between 6 weeks – 4.5 years (133–136). The triad of epilepsy (usually gelastic (laughing) or dacrystic (crying) seizures), central precocious puberty, and developmental delay is classic with the seizure semiology eventually evolving into multiple, more severe seizure types (130). Rarely, they are associated with Pallister-Hall syndrome, an autosomal dominant disorder characterized by polydactyly and other midline defects (imperforate anus, bifid epiglottis, panhypopituitarism and dysmorphic facies) (132,137), or with SOX2 mutations (138).

The intrinsic epileptogenicity of these lesions (139,140), the trend towards evolving seizure semiology, the worsening of behavioral and psychiatric co-morbidities, and the general failure of anti-epileptic drug therapy has led clinicians to explore the options of surgical or stereotactic radiosurgical resection despite their deep-seated location, with variably reported success in the remission of seizure activity and behavioral disturbances, but more modest improvements in cognitive function (130,131,141–143). Li et al.'s (144) case series reported successful remission of central precocious puberty (CPP) and little, if any, late-onset endocrinopathy; but a larger cohort study by Freeman et al. (145) suggested that clinically silent endocrine dysfunction (particularly GH and TSH deficiency) is common both at diagnosis and postoperatively. Transient posterior pituitary dysfunction leading to DI and the syndrome of inappropriate antidiuretic hormone secretion (SIADH) has also been described (145,146). One adult cohort study corroborates these findings, showing that >1/3 of these patients had endocrine dysfunction and approximately 2/3 experienced excessive weight gain postoperatively (147). More recently laser induced thermal therapy (LiTT) of these lesions has shown promising results with regards to seizure control, with little late onset additional endocrinopathies (148,149).

Langerhans Cell Histiocytosis (LCH)

Figure 6. T₁-weighted MRI image demonstrating the appearances of a contrast-enhancing suprasellar LCH lesion. There is a small anterior pituitary and absent posterior pituitary bright spot in keeping with the known panhypopituitarism (including central DI) present at diagnosis.

LCH (previously “histiocytosis X”) is one of the three major histiocyte disorders, and involves clonal proliferation of bone marrow-derived dendritic antigen-presenting (“Langerhans”) cells which accumulate in various organs (150). It is a rare disease with an incidence of 2.6-8.9 cases/million/year, the majority presenting in infancy (median age at diagnosis 2-3.8 years, incidence at age <1 year 9.0-15.3 cases/million/year vs. age >5 years 0.7-4.5 cases/million/year) with no sex predilection (151–154). The variability in organ involvement causes a spectrum of clinical features ranging from a single self-healing cutaneous lesion to fatal multiorgan disease, particularly if the liver, spleen, lungs, and hemopoietic system (the “risk” organs) are involved (150). Multisystem involvement is present in 27-56% of cases, of which 28-80% have “risk” organ involvement (151–153,155,156). LCH can thus be considered a primary hematological disorder which, in a proportion of cases, infiltrates the CNS, although its etiology, whether neoplastic or reactive, remain poorly understood (155). More than half of biopsied lesions contain BRAF mutations(157).

In the CNS, the hypothalamo-pituitary region is involved in up to 25% of cases, which almost invariably leads to DI (previously known as Hand-Schuller-Christian disease if associated with orbital and bony lesions)(151,152,154,158,159). Commonly associated radiological findings include thickening of the pituitary stalk with progression to space-occupying tumors and an absence of the posterior pituitary bright spot (159). Indeed, LCH is the commonest underlying diagnosis in patients with central DI and an intracranial mass, occurring in 70% of this cohort(160). The presence of multisystem involvement, particularly if involving “risk” organs, craniofacial bones, gastrointestinal tract, skin, or genitalia) is a particular risk factor for DI (159,161).

Treatment is dependent on the number of organs involved and may range from biopsy/curettage, intralesional steroids, indomethacin, and radiotherapy/UV phototherapy for single bone and cutaneous lesions to systemic chemotherapy with steroids and vinblastine for multisystem disease (155,162,163). Refractory cases have been treated with cytarabine, cladribine, clofarabine, hemopoietic stem cell transplantation, or BRAF inhibitors (164–168). Notably, no treatment protocol has been shown to reverse existing or prevent future DI or other endocrinopathies(159), though current therapeutic recommendations are aimed at preventing disease progression and limiting endocrinopathy with prolonged, low-dose systemic chemotherapy (155,169–171). Overall, 5-year survival remains relatively high at 71-95%, but 3-25% of patients experience at least one endocrinopathy (particularly GH deficiency), with no current chemotherapeutic regimens showing superior overall- or endocrine event-free survival (151,156,158,161).

Pituitary Stalk Thickening

Figure 7. T₁-weighted MRI image illustrating the appearances of a contrast-enhancing thickened pituitary stalk lesion (arrow) and an absent posterior pituitary bright spot in a patient presenting with central DI. The differential diagnosis included germinoma and LCH. However, approximately one year after diagnosis, the pituitary stalk lesion resolved completely, although the patient has been left with GH deficiency and central DI.

A thickened pituitary stalk (TPS) may be discovered either as part of the evaluation of a patient presenting with central DI, visual impairment, or other endocrine dysfunction or incidentally on neuroimaging performed for other purposes. It is discussed here as it is an important differential for germ cell tumors and Langerhans cell histiocytosis (LCH), resulting frequently in diagnostic and management dilemmas, due to a number of reasons:

There is no clear consensus as to what constitutes abnormality for children; previous adult studies have shown that the 95^th centile for the transverse dimensions of the infundibulum at the optic chiasm and pituitary insertion are 4.21-4.35 mm and 2.69-2.89 mm respectively (upper limit 4.21-4.58 mm and 2.93-3.04 mm) (172,173). Raybaud and Barkovich suggest using a pediatric threshold thickness of 3.8 mm at the optic chiasm and 2.7 mm at the pituitary insertion for investigating further pathology, particularly if there are interruptions in the normal smooth tapering of the infundibulum from median eminence to pituitary insertion (174).
The radiological appearances of a TPS, LCH and germinomas cannot be easily differentiated and there is substantial overlap (Table 2). The normal infundibulum lacks a blood-brain barrier and therefore always enhances with contrast, obscuring neoplastic processes. TPS is the commonest initial radiological finding in both LCH and germinomas, and concurrent absence of the posterior pituitary bright spot is inconsistent (123,175,176). Similarly, the two commonest causes of TPS in the pediatric age group are LCH and germinomas, accounting for 7-75% and 9-71% of TPS cases respectively (176–179). Other common causes of TPS in adults such as lymphocytic hypophysitis and neurosarcoidosis are rare in children (176).
Biopsies of the TPS to obtain a definitive histological diagnosis can be inconclusive and lead to further substantial endocrine morbidity, including panhypopituitarism with DI, and are thus generally avoided (178).
The interval from the time of initial symptoms to diagnostic MRI can be prolonged, particularly for germinomas (up to 21 years), occasionally with initially normal neuroimaging (123,124,180,181). An initially normal MRI does not therefore preclude an occult germinoma or other pathological process in the presence of idiopathic central DI, leading some authors to recommend serial 3-6 monthly scans and follow-up, although the duration of serial scanning is unclear (174). Additionally, there have been cases of occult germinomas masquerading as radiologically or even histologically diagnosed lymphocytic hypophysitis in children (182,183).

In an attempt to define which patients with isolated TPS are at risk of neoplasia and therefore require more intensive follow-up or biopsy, Robison et al. suggest risk factors such as the presence of DI (strongest risk factor), the coexistence of DI with anterior pituitary dysfunction or a progressive increase in infundibular size of >15% from baseline (178). Apart from size, no other particular MRI appearances have been found to be specific for pediatric-related tumor processes (184). Various proposed diagnostic pathways have been proposed for the management of TPS and idiopathic DI (178,184,185) but most recently a national consensus-based guideline has been developed in the UK by the CCLG, RCPCH and BSPED to help achieve a more consistent approach to this finding (186).

Miscellaneous Non-Neoplastic Hypothalamo-Pituitary Masses

Other hypothalamo-pituitary malformations can mimic neoplastic processes in the suprasellar region, and should therefore be considered in the differential diagnosis particularly before commencing oncological therapies:

Pituitary hyperplasia – Hypothalamic releasing hormones are trophic on the pituitary gland, hence hypersecretion of these hormones (e.g., GHRH from a pancreatic tumor in children with MEN1 syndrome) can cause anterior pituitary enlargement and mimic a true mass. The commonest physiological cause of pituitary hyperplasia is puberty, where the maximal height of the gland can be 10 mm in girls and 7 mm in boys (187,188). Pituitary hyperplasia can also occur pathologically, for instance in chronic primary hypothyroidism leading to thyrotroph hyperplasia due to a lack of negative feedback (24,187). It is also important to note that pituitary enlargement can be associated with certain congenital forms of hypopituitarism (PROP1, LHX3, SOX3 mutations (189,190).
Rathke’s cleft cysts (RCCs) – RCCs are congenital cystic epithelial remnants of Rathke’s pouch which fail to involute during pituitary development, hence arising in the pars intermedia but often extending superiorly (24). Although often incidental and asymptomatic (occurring in 11% of autopsy cases (191)), cystic growth can lead to visual deficits and endocrinopathies, requiring surgical marsupialization (resection exacerbates endocrine dysfunction) (192). Unlike craniopharyngiomas (the other common cystic suprasellar lesion), RCCs do not calcify.
Arachnoid cysts – These are congenital collections of cerebrospinal fluid (CSF) arising from the splitting and/ or duplication of the arachnoid membranes. 16% are suprasellar and these cysts can present with symptoms of raised intracranial pressure, visual deterioration, endocrinopathies, or developmental delay (193–197). Treatment is by endoscopic fenestration (196,198,199).
Rare entities – In contrast to adults where autoimmune lymphocytic hypophysitis is the commonest cause of isolated thickened pituitary stalk (TPS), this is exceptionally rare in children, but should be considered in the differential together with other granulomatous diseases (neurosarcoidosis, tuberculosis (24,200).

Table 2. The Differential Diagnosis of Pediatric Suprasellar Masses by Radiological Features
Tumor	Primary location	T1 intensity^§	T2 intensity^§	Contrast enhancement	Other features
Craniopharyngioma	Supra>intrasellar	Variable, heterogenous	High	Yes (cystic rims)	Cysts, heterogenous, calcification
LGG	Suprasellar, optic pathways	Low	High	Yes	Generally homogenous
Pituitary adenoma	Intrasellar (intrapituitary)	Low	Low	No	Sella turcica expansion
Germinoma*	Suprasellar, pituitary stalk	Isointense – low	Isointense – low	Yes	Loss of posterior pituitary bright spot, coexistent pineal tumor
Hamartoma	Suprasellar (tuber cinereum)	Isointense	Isointense – high	No	-
LCH*	Suprasellar, pituitary stalk	Isointense	Isointense	Yes	Loss of posterior pituitary bright spot, coexistent osseous lesions
Lymphocytic hypophysitis*	Suprasellar, pituitary stalk, intrasellar	Isointense	Isointense	Yes	Loss of posterior pituitary bright spot
Pituitary hyperplasia	Intrasellar	Isointense	Isointense	Yes	Homogenous
RCC	Intrasellar	Isointense – high	Isointense – low	No	Round & smooth walled
Granuloma (sarcoidosis, TB)	Suprasellar, pituitary stalk	Isointense – low	Low – isointense	Yes	Coexistent parenchymal and leptomeningeal lesions
Arachnoid cyst	Suprasellar	Very low (isointense with CSF)	High (isointense with CSF)	No	-

LGG, low-grade glioma; LCH, Langerhans cell histiocytosis; RCC, Rathke’s cleft cysts. §MRI signal intensity in comparison to that of gray matter. *Note that germinomas, LCH and lymphocytic hypophysitis cannot be differentiated on radiological features alone (24,26,174,201).

NEUROENDOCRINE DYSFUNCTION AT DIAGNOSIS OF HYPOTHALAMO-PITUITARY TUMORS

Neurological Syndromes

RAISED INTRACRANIAL PRESSURE (RICP)

The proximity of hypothalamo-pituitary tumors to the floor of the third ventricle and optic chiasm accounts for the high frequency of RICP and visual symptoms at presentation. RICP symptoms (headache, vomiting, and/or papilloedema) are the commonest presenting feature of any pediatric brain tumor (30-60%) (202,203), but occur with even greater frequency in suprasellar lesions such as craniopharyngiomas (78%) and LGGs (86%) (37,66). Children may therefore present to acute neurosurgical units as a neurosurgical emergency or subacutely with a chronic course that may initially be misdiagnosed as tension/ migrainous headaches or infective gastroenteritis with unrecognized concurrent visual disturbances. Current UK recommendations are to scan all children with vomiting persisting <2 weeks, and/ or headaches occurring in children <4 years, on waking or during sleep, in association with confusion and/ or disorientation, or persisting >4 weeks (9). Persistent vomiting in the absence of other features suggestive of gastroenteritis (diarrhea, pyrexia) should also prompt consideration of an intracranial lesion. It is important to note that due to the delayed fusion of cranial sutures, children <4 years of age with hydrocephalus more often (41%) present with a rapidly increasing head circumference than classical RICP symptoms (203).

VISUAL DETERIORATION

Visual field loss and/or worsening visual acuity are the second commonest presenting feature, particularly in LGGs, where up to 100% of cases may have visual impairment due to direct involvement of the optic pathway (75). Other suprasellar tumors affect visual function by mass effect on the optic chiasm, occurring in up to 50-70% of craniopharyngiomas and 15% of pituitary adenomas (38,44,102). Contrastingly, visual symptoms are rare (~5-7%) in children with other CNS tumors (203). Other common ophthalmological symptoms that warrant urgent neuroimaging include new onset nystagmus, incomitant (paralytic) squints, optic atrophy, and proptosis, particularly given the difficulties in assessing visual function in young children and the danger of passing off a squint as being “normal” in childhood without detailed examination (9,203,204). Parinaud’s syndrome, a combination of upward gaze palsy, convergence-retraction nystagmus, and pupillary dilatation with light-near dissociation is a rare particular presentation of bifocal suprasellar/pineal germinomas due to pressure of the pineal tumor on the tectal plate (124,205). Although the aim of oncological therapy in many of these low-grade tumors is to preserve vision, this has not been generally successful, most likely due to nerve fiber dropout and optic atrophy (84), or the fact that anatomical tumor characteristics correlate poorly with the degree of visual loss at diagnosis (206).

SEIZURES

Seizures are an uncommon presenting clinical feature of pediatric hypothalamo-pituitary tumors, occurring in <10% of craniopharyngiomas, LGGs, and germinomas (35,39,124,207,208), and are more often the result of reversible metabolic causes such as hypoglycemia (from cortisol and/or GH insufficiency), hypernatremia (from DI), or hyponatremia (from SIADH). Gelastic or dacrystic (laughing or crying, from the Greek gelos and dakryon respectively) seizures are notoriously difficult to diagnose but are characteristic of hypothalamic hamartomas (80-90%) due to the intrinsic epileptogenicity of these lesions that are essentially disorders of neuronal migration (134,139,147).

OTHER NEUROLOGICAL AND COGNITIVE SYMPTOMS

Hemiparesis and ataxia are less common but significant presenting features of intracranial tumors, as are cognitive impairment, delayed development, behavioral changes, and psychiatric symptoms, all of which mandate detailed neuro-ophthalmological examination in such cases, particularly in the presence of the neurocutaneous stigmata of tumor-predisposing syndromes such as neurofibromatosis and tuberous sclerosis.

Endocrine Dysfunction

Although neuro-ophthalmological symptoms are the commonest presenting feature of hypothalamo-pituitary lesions, they are often preceded by symptoms associated with undiagnosed endocrinopathies in as many as two-thirds of patients (209). Endocrine dysfunction may be due to hormone excess (e.g., secreting pituitary adenomas, central precocious puberty) or hormone deficiency from pituitary invasion or compression by tumor mass, disrupting the various hypothalamo-pituitary endocrine pathways. The incidence of dysfunction in each of the hypothalamo-pituitary axes is partly dependent on the lesion (Table 3) though the reasons for the specificity of these presentations are largely unknown.

GH deficiency (GHD) and gonadotrophin dysfunction (either central precocious puberty (CPP) or gonadotrophin deficiency (GnD, i.e., pubertal delay/arrest)) are often the initial and commonest endocrinopathies at presentation of both craniopharyngiomas (GHD – up to 100%; GnD – up to 85%, CPP – up to 3%) and LGGs (CPP – up to 56%; GHD – up to 27%; GnD – up to 12%) (37,41,42,66,210). CPP is particularly prevalent in LGGs as it can occur in the context of NF-1 even in the absence of a hypothalamo-pituitary lesion (211). It is also one of key components of the hypothalamic hamartoma clinical triad, present in up to 45% of patients at diagnosis (131,145). In both these cases it is presumed to result from premature activation of hypothalamic GnRH, unlike its occurrence in up to 35% of germinomas, where gonadotrophin-independent CPP can occur due to secretion of β-hCG which shares a common alpha subunit with LH and FSH and thus stimulates the same receptors (124,126).

Other anterior pituitary deficits evolve only with extensive disease, and are usually only seen at presentation with craniopharyngiomas, although more subtle deficits may have previously been under-recognized with other tumors. ACTH deficiency (secondary hypoadrenalism) is particularly important to diagnose and treat pre-operatively, and is present at diagnosis in up to 71% of craniopharyngiomas, 19% of germinomas, 10% of hamartomas and 3% of LGGs (41,124,145,212). Similarly, TSH/TRH deficiency (secondary/central hypothyroidism) is present in up to 32% of craniopharyngiomas, 19% of germinomas and 10% of LGGs and hamartomas(45,124,145,213). Mild to moderate hyperprolactinemia (<2000 mU/l) is common in all non-prolactinoma hypothalamo-pituitary lesions, needs to be distinguished from true prolactinomas (>5000 mU/l), and does not usually lead to clinically significant galactorrhea.

Posterior pituitary dysfunction, particularly central (“cranial”) DI, is the hallmark endocrinopathy of germinomas and Langerhans cell histiocytosis (LCH), being present in up to 90% and 40% of patients respectively at diagnosis(123,158). However, DI can also occur as a presenting clinical feature for other suprasellar lesions which may be missed if symptoms of polyuria and polydipsia are not elucidated.

Table 3. Common Endocrinopathies at Presentation of Various Hypothalamo-Pituitary Lesions
Tumor	Commonest endocrinopathy at presentation
Craniopharyngioma	GH deficiency, pubertal delay/arrest
Optic pathway LGG	Central precocious puberty
Pituitary adenoma	Hyperprolactinemia (prolactinomas)
Suprasellar germinoma	Central diabetes insipidus, gonadotrophin-independent central precocious puberty (hCG-secreting)
Hypothalamic hamartoma	Central precocious puberty
Langerhans cell histiocytosis	Central diabetes insipidus

GH, growth hormone; LGG, low-grade glioma; hCG, human chorionic gonadotrophin.

Endocrine dysfunction is under-recognized at presentation, as demonstrated by the discrepancies between spontaneous reports of growth retardation, weight loss/gain, polyuria and polydipsia compared to their true incidence based on direct enquiry or assessment (44). Longitudinal retrospective studies have shown that growth failure and weight gain can occur up to 3 years before the diagnosis of a craniopharyngioma, especially in the presence of hypothalamic infiltration (214). Since the diagnosis of GH deficiency requires dynamic endocrine testing, and idiopathic CPP can be a normal variant in young girls, a significant underlying lesion may be missed without mandatory neuroimaging, despite studies showing that 14-45% of female patients with CPP have a hypothalamo-pituitary mass (215–217). DI may remain occult in the ACTH-deficient patient, or unrecognized until the patient is water-deprived or rendered effectively adipsic by general anesthesia, coma or further hypothalamic damage sustained during surgery, with potentially fatal consequences. Lethargy, recurrent infections, somnolence, and cold intolerance may be subtle symptoms of ACTH and/or TSH deficiencies, whilst hypothalamic dysfunction (discussed below) manifesting as hyperphagia, escalating obesity, sleep-wake cycle disturbance, and temperature dysregulation may be mistaken for psychosocial dysfunction.

PRE-OPERATIVE ENDOCRINE ASSESSMENT AND MANAGEMENT OF HYPOTHALAMO-PITUITARY TUMORS

Due to their relative rarity and a general lack of data on optimum treatment strategies, all pediatric hypothalamo-pituitary tumors should be discussed in a multidisciplinary forum which comprises, at minimum, a neuro-oncologist, neuroradiologist, pediatric endocrinologist, and pituitary surgeon. Careful endocrine assessment with appropriate neuroimaging is vital before definitive therapy (Table 4). Early morning cortisol/ACTH measurements should ideally be performed before any dexamethasone is given for cerebral oedema, alongside paired urine and plasma osmolarities & electrolytes as these will influence perioperative fluid management. Plasma tumor markers (prolactin, β-hCG, α-fetoprotein) should be obtained prior to any surgical intervention regardless of radiological appearances, as both prolactinomas and germinomas can be treated medically without requiring a biopsy. In some cases, cerebrospinal fluid β-hCG and α-fetoprotein may be required to aid diagnosis. Early access to a pediatric endocrinologist enhances diagnostic decision-making and ensures appropriate peri-operative fluid management particularly in the presence of life-threatening salt/water and hypocortisolemic crises. If dexamethasone has not been commenced for peritumoral edema and where a patient’s hypothalamo-pituitary-adrenal status is unknown, parenteral hydrocortisone (2 mg/kg) should be given at anesthetic induction and 6-8 hourly thereafter for 48-72 hours (or via a continuous hydrocortisone infusion), weaning to maintenance doses over 5-10 days according to clinical status until this axis can be formally assessed with a synacthen test. Clinicians should be aware of cortisol’s permissive effects on the renal tubule for free water clearance; thus, its replacement will unmask occult DI. In this situation, precise fluid balance measurements and the judicious use of desmopressin by an experienced endocrinologist are required. GH, thyroxine and estradiol/ testosterone supplementation may also be necessary. It is important to note that thyroid hormone replacement should not be commenced until a patient is cortisol replete for at least 48 hours to avoid precipitating an adrenal crisis.

Table 4. Recommended Minimum Pre-Treatment Endocrine Assessment for Hypothalamo-Pituitary Tumors

Clinical assessment

Height

Weight

Sitting height

BMI

Tanner pubertal stage

Bone age

Endocrine biochemistry

IGF-1/IGF-BP3

LH, FSH, estradiol/testosterone

TSH, free T4 ± free T3

Early morning (8-9 am) cortisol & ACTH

Early morning paired urine & plasma osmolarities & electrolytes

Tumor markers

PRL

AFP

β-hCG

BMI, body mass index; IGF-1, insulin-like growth factor 1; IGF-BP3, insulin-like growth factor binding protein 3; LH, luteinizing hormone; FSH, follicle-stimulating hormone; TSH, thyroid stimulating hormone; T₄, thyroxine; T₃, triiodothyronine; ACTH, adrenocorticotrophic hormone; PRL, prolactin; AFP, alpha-fetoprotein; β-hCG, beta-human chorionic gonadotrophin.

Rare Emaciation/Failure To Thrive Syndromes

DIENCEPHALIC SYNDROME (DS)

DS is a rare syndrome of severe emaciation first described over 60 years ago typically seen in infants <2 years of age in the presence of a hypothalamic tumor (218). The original description incorporated four “major” criteria – profound emaciation (often leading to a multitude of misdirected investigations for failure to thrive), preserved (or accelerated) linear growth, hyperactivity, and euphoria – and three “minor” features: pallor without anemia, hypoglycemia, and hypotension. There is marked loss of subcutaneous fat despite increased caloric intake. Other associated features result from either tumor location (nystagmus, papilloedema, optic atrophy, vomiting, ataxia) or increased sympathetic tone (sweatiness, tremor). Classically, DS occurs in <10% of hypothalamic LGGs (11,209), but has also been described in suprasellar high grade gliomas (77,219), germinomas (220,221), teratomas (222), ependymomas (223), craniopharyngiomas (224), epidermoid cysts (225), and rarely with non-suprasellar lesions such as brainstem gliomas(226). Since Russell’s original description, however, the definition for DS has now too loosely broadened to include all cancer-related cachexia (227), with <4% of patients with DS having onset of symptoms at >2 years of age (220,228), and some publications reporting adult-onset DS where growth velocity is irrelevant (224,229). It is therefore becoming increasingly difficult to determine whether the patients described in these cases truly have DS or not. Its pathophysiology remains poorly understood, although the most consistent biochemical finding is of high random plasma GH concentrations that is neither suppressed by an oral glucose tolerance test, nor further stimulated by insulin-induced hypoglycemia, with low or normal IGF-1 concentrations, indicative of a GH-resistant state(77,230,231). Studies showing increased resting energy expenditure (232,233) support the theory of a dysregulated metabolism rather than abnormal caloric intake. At the time of writing, the next LGG trial is being designed to incorporate an international study of this rare entity, which is an independent risk factor for death, progression (11) and severe endocrine morbidity (25).

ANOREXIA AND EATING DISORDERS

Anorexia nervosa is an over-represented symptom in multiple published case reports of patients with hypothalamic lesions (particularly slow-growing germ cell tumors), with an average delay in diagnosis of nearly 3 years (234), though symptoms tend to resolve with appropriate therapy. Given the ventromedial and lateral hypothalamic location of the hunger and satiety centers, it is reasonable to postulate the effect of a suprasellar lesion on appetite. However, current understanding of the orexigenic and anorexigenic neuroendocrine regulators of tumor-related anorexia is still incomplete, and reports of non-suprasellar CNS tumors presenting with anorexia (227,235,236) suggest dysregulation beyond the hypothalamus, whilst the effect of inflammatory cytokines present in disseminated disease (tumor necrosis factor-α (TNF- α), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-γ (IFN- γ)), may also play a role (227). An intracranial lesion needs to be differentiated from true anorexia nervosa, which should fulfil DSM-V or ICD-10 criteria(237,238)), in all patients presenting with anorexia and weight loss. A full auxological, pubertal and endocrine biochemical assessment should be performed to exclude neuroendocrine disease, particularly in boys where the lower prevalence of anorexia nervosa requires mandatory pituitary neuroimaging. Anorexia nervosa presenting with amenorrhea may be due to a suprasellar tumor causing hypogonadotrophic hypogonadism (239), and initially normal imaging may not exclude an eventual diagnosis of a tumor, particularly for germinomas (235). Severe weight loss at diagnosis may be a predictor for future hypothalamic obesity (240).

NEUROENDOCRINE DYSFUNCTION AFTER DIAGNOSIS AND/OR TREATMENT

The Evolution Of Endocrinopathy And Its Association With Treatment

Whilst the initial endocrinopathies present at diagnosis are fairly typical for particular tumor subtypes, the pattern of post-treatment endocrine dysfunction in survivors of these lesions is interestingly very similar in frequency and timing. It has long been recognized that there is an evolution in the incidence of dysfunction in each of the hypothalamo-pituitary axes over time, closely mimicking that seen in congenital neurodevelopmental disorders such as septo-optic dysplasia (241). Although the various axes are differentially sensitive to irradiation, with the GH axis being the most susceptible (even at doses as low as 20 Gy), and the ACTH axis being the most robust (38,242,243), the similar evolutionary pattern of endocrine dysfunction seen in patients with a wide range of hypothalamo-pituitary lesions even in the absence of therapeutic irradiation suggests that the pattern of deficits is related most strongly to the position of the tumor (and thus recurrent disease) rather than treatment. GH deficiency is thus commonest, followed by gonadotrophin dysfunction (either central precocious puberty or hypogonadotrophic hypogonadism), ACTH and TSH deficiency, and least commonly posterior pituitary dysfunction, usually presenting as central DI (which is never seen after similar pituitary irradiation doses administered to non-suprasellar tumors) (25,37,45,145,158,244–247). Hence, lifelong endocrine follow-up of these survivors with regular clinical and biochemical assessments is vital as all patients with such tumors remain at high-risk for the development of these deficits. National guidelines on the neuroendocrine long-term follow-up of tumors such as craniopharyngiomas have been developed in the UK (49).

GH Deficiency

GH deficiency affects virtually all survivors of pediatric hypothalamo-pituitary lesions at some stage. If not already present at diagnosis, it is virtually guaranteed to occur after pituitary-directed therapy such as radiotherapy or surgery(45,248). Diagnosis of GH deficiency requires dynamic endocrine testing with the gold standard being the insulin tolerance test, although this is contraindicated in patients with a history of seizures. It is worth noting that the GHRH stimulation test should not be used in this context as it will not detect GH deficiency of hypothalamic origin (249). Serum IGF-1 and its binding protein IGF-BP3 are less accurate markers of GH deficiency, although they may be useful in severe growth failure in the context of a hypothalamo-pituitary tumor where GH testing is considered too hazardous (250,251). They should not be used in the context of suspected GH deficiency in the context of previous irradiation (252–254). Occasionally, GH deficiency may initially present with abnormal spontaneous secretion but normal peak responses to stimulation tests (termed “neurosecretory dysfunction”) (255), although testing for this with overnight GH profiling is not currently recommended by the GH Research Society (256).

Paradoxical normal growth may continue despite GH deficiency either due to precocious or accelerated puberty, or the syndrome of “growth without growth hormone”, where secondary hyperinsulinemia occurs due to the rapid weight gain observed post-treatment (257). Growth failure may also be masked by concurrent central precocious puberty. Both situations deserve prompt investigation and GH substitution which, in replacement doses, does not increase tumor recurrence (25,258–260), but promotes anabolism and lean body mass. This should therefore not be delayed beyond 12 months after definitive therapy (although this cut-off is arbitrary) (261), particularly in patients who have irreversible loss of height from spinal irradiation (e.g., for germinomas) (262).

Gonadotrophin Dysfunction

Gonadotrophin dysfunction may manifest in three ways. Firstly, central precocious puberty (CPP) (defined as a testicular volume of ≥4 ml in a boy <9 years or breast budding in a girl <8 years) which, if not already present at diagnosis (e.g., hamartomas, LGGs, germinomas) is increased particularly by radiotherapy (243). There is no evidence that surgical resection of hypothalamic hamartomas, the commonest lesion associated with CPP, improves these symptoms, despite ameliorating the seizures (145). As mentioned above, coexistence of an early puberty with GH deficiency may mask the latter as height velocity may initially appear to be maintained or even accelerated, but not when corrected for bone age. Any child in puberty should therefore concurrently have an urgent assessment of GH secretion and consideration of replacement to restore height in combination with GnRH analogues to delay skeletal maturation if it is felt psychosocially appropriate. It is worth noting that prior CPP does not preclude later pubertal delay or arrest and may in fact be a risk factor (25). Therefore, careful monitoring is required even after the cessation of GnRH analogues.

Pubertal delay or arrest may either be due to hypogonadotrophic hypogonadism from tumor- or treatment-related injury to the hypothalamus (causing GnRH and/or LH/FSH deficiency) or to primary gonadal failure from systemic chemotherapy (hypergonadotrophic hypogonadism). Patients may fail to enter puberty altogether by the expected age (14 years in boys, 13 years in girls), enter puberty normally and subsequently fail to progress, or demonstrate secondary amenorrhea (girls) or sexual dysfunction (boys). In this situation concurrent GH deficiency can be corrected simultaneously or 6 months prior to commencing sex steroid replacement to initiate an appropriately-timed pubertal growth spurt. There is no advantage to adult height in delaying sex steroid replacement any further, particularly in the light of the benefits on bone mineral accretion (263).

Most chemotherapeutic drugs used in CNS tumor regimens (e.g., carboplatin, vincristine, etoposide) are not considered gonadotoxic, but other high-risk agents such as cyclophosphamide, temozolomide, and cisplatin are occasionally used, with their effects being modulated by age at exposure and gender (264). Since it is possible to protect future fertility in boys even as young as 12 years with some masculinization (Tanner stage 3+ and/or testicular volume of 8+ mls) by sperm cryopreservation, this should be considered before definitive therapy, even in those not receiving chemotherapy (265). By contrast, girls who have achieved regular spontaneous menses should be warned of the reduced window of reproductive capacity and a premature menopause due to a reduced ovarian follicular reserve (266). Notably, patients with hypothalamo-pituitary tumors who have received chemotherapy can potentially have concurrent hypogonadotrophic hypogonadism and primary gonadal failure, compounding the future risk of subfertility.

ACTH Deficiency/Central Adrenal Insufficiency

The hypothalamo-pituitary-adrenal (HPA) axis is fortunately relatively robust to irradiation and chemotherapeutic damage. However, in the context of a hypothalamo-pituitary tumor, the most important diagnostic challenge is to accurately determine adrenal reserve and differentiate reversible dexamethasone-induced ACTH suppression (after treatment for cerebral edema) from true, permanent ACTH deficiency. Given the lifelong implications of the latter, it is our opinion that the diagnosis should be carefully made ideally with the gold standard insulin tolerance test (ITT) and repeatedly reviewed with time. This may additionally necessitate regular plasma morning cortisol and ACTH measurements and 24-hour cortisol day curves. Although the standard synacthen test (SST) is often used to test adrenal integrity in adults, this supraphysiological stimulus does not test the entire pathway and the integrity of the hypothalamus or pituitary. There is evidence to suggest that in CNS tumor survivors the SST may be less sensitive than the ITT or low dose synacthen stimulation in detecting more subtle degrees of deficiency (267–269). In patients who have received peri-operative dexamethasone for peritumoral edema, formal testing of the HPA axis may be best left until 2-3 months after substitution with maintenance hydrocortisone as doses can be more safely omitted whilst testing is performed. Testing should be performed in a tertiary pediatric endocrinology unit used to managing patients with multiple endocrinopathies, with routine glucose rescue at 25-30 minutes and hydrocortisone at the end of low-dose (0.1 units/kg) insulin-induced hypoglycemia or glucagon stimulation. Treatment of adrenal insufficiency with glucocorticoids may unmask occult DI, and the coexistence of ACTH deficiency, DI, and adipsia due to hypothalamic damage can be fatal and should be avoided where possible.

TRH/TSH Deficiency/Central Hypothyroidism

The thyroid, like the hypothalamo-pituitary-gonadal axis, can be rendered underactive by either central TRH/TSH deficiency (inappropriately normal/low TSH for a low free T₄ or T₃) due to the tumor itself or surgery, or primary hypothyroidism (high TSH with a normal (compensated/subclinical) or low (frank) free T₄) from spinal irradiation and/or chemotherapy, with the potential for the two states coexisting in some patients. There is little evidence for the role of irradiation in the former. In the adult cohort studied by Littley et al., no patients treated with low-dose radiotherapy alone experienced TSH deficiency (242). Similarly, Gan et al. found that the only independent risk factor for TSH deficiency in LGGs was hypothalamic involvement of the tumor (25). TRH stimulation tests may not differentiate hypothalamic (tertiary) from pituitary (secondary) damage, and serial thyroid function tests with two consecutive low free T₄ concentrations in association with a low or inappropriately normal TSH concentration confirm the diagnosis without the need for further testing (270–272).

Primary hypothyroidism can present many years after the initial irradiation or chemotherapeutic insult. Annual thyroid function tests in at-risk children are important for early detection of subclinical hypothyroidism and institution of early treatment, particularly in light of the known effects on the developing brain. Given the known risk of radiation-associated second primary thyroid cancers, the carcinogenicity of nuclear fallouts, and the long-term cardiovascular mortality risk of subclinical hypothyroidism, few clinicians would leave a persistently raised TSH in such a patient cohort untreated (273).

Hyperprolactinemia

The importance of serum prolactin (PRL) measurements in the diagnosis of prolactinomas has already been discussed. Similarly, a rise in PRL levels can occur post-treatment in two situations. In the presence of a prolactinoma, this can indicate tumor “escape” from dopamine agonist (cabergoline/bromocriptine) control requiring further therapy. The more common situation arises where hyperprolactinemia is due to stalk compression by a progressive sellar or suprasellar tumor or hypothalamic damage. In this situation PRL concentrations are usually <2000 mU/l (274) and patients are unlikely to be symptomatic, with galactorrhea being unusual(25). Chronic severe primary hypothyroidism will also lead to hyperprolactinemia due to the stimulatory effects of a raised TRH on the lactotroph.

Posterior Pituitary Dysfunction (PPD)

Posterior pituitary dysfunction can present itself in three ways – DI, SIADH, or cerebral salt-wasting syndrome (CSW), the latter attributed to hypersecretion of cerebral atrial natriuretic (ANP) and brain natriuretic peptides (BNP) in response to plasma volume expansion by ADH. The latter two syndromes are rare outside the context of an acute cerebral insult and are usually transient, whilst DI may be a presenting feature and/or a permanent post-operative deficit. The absence of a posterior pituitary bright spot on MRI is a relatively sensitive marker of a lack of neurohypophyseal integrity (275–277). DI does not develop after cranial irradiation in the absence of a hypothalamo-pituitary tumor or surgery to the area (25,99). Whilst PPD is the least common form of endocrinopathy, the rapid shifts from hyper- to hyponatremia in the acute setting can prove life-threatening, as evidenced by a recent retrospective cohort study of optic pathway LGGs with high survival showing showed that nearly 50% of the deaths that occurred were associated with uncontrolled PPD (25). This risk is further increased by coexistent ACTH deficiency, hypothalamic adipsia, and treatment with anti-epileptic medications, which have SIADH-like effects.

After hypothalamo-pituitary surgery, PPD presents as a well-described triphasic response in ADH secretion: firstly, immediate but transient DI up to day 2; secondly, SIADH from day 1-14; and finally, a second phase of DI, which is usually permanent if it persists beyond 21 days, the preceding SIADH is prolonged or severe, or if extensive surgery has been performed (278,279). This triphasic response is thought to result from necrosis of hypothalamic ADH-secreting magnocellular neurons and is seen more often in children than adults (23% vs. 14% in one craniopharyngioma study) (280). The three phases may also occur independently, and cerebral salt-wasting syndrome may coexist and complicate diagnosis and management. Dramatic changes in sodium concentrations can therefore occur with the inherent risk of seizures, cerebral edema and death; such patients require high intensity care with precise fluid management supervised by an experienced pediatric endocrinologist. The measurement of plasma and urinary arginine-vasopressin (AVP) may help differentiate between these different disorders, but these assays are not widely available and careful sample processing is required prior to analysis (281). More recently, measurement of plasma copeptin, the more stable by-product of cleavage of AVP from its carrier protein neurophysin II, is becoming more widely available and has been shown to be a more easily measurable, sensitive, and specific surrogate marker of AVP secretion (282–284).

Detailed management of these disorders is beyond the scope of this chapter, but can be summarized in the algorithm seen in Figure 8.

Figure 8. Algorithm for the management of post-operative salt-water balance disorders (53).

The Hypothalamic Syndrome

The hypothalamic “syndrome” is loosely defined and usually refers to a constellation of features attributed to hypothalamic dysfunction. Central to it is hypothalamic obesity, a morbid, inexorably escalating obesity (BMI usually >+3 SDS) first described over a century ago (285). It occurs in up to 77% of craniopharyngiomas, 53% of optic pathway LGGs, 40% of pituitary adenomas, 40% of germinomas, and 23% of hamartomas (64,145,286–288), with some symptoms occurring at diagnosis prior to any treatment. Despite this, its pathophysiology is still poorly understood, although it is becoming increasingly evident that both hyperphagia and a dysregulation of anorexigenic and orexigenic hormones contribute (289). Young age at diagnosis, hypothalamic injury by tumor, high dose irradiation or surgery (including biopsies), and multiple endocrinopathies are all risk factors (278,289). Unlike common obesity, the weight gain is largely resistant to caloric restriction, lifestyle interventions, medical and surgical therapies (290–295). Several authors have recently trialed GLP-1 agonists in hypothalamic obesity with some success (296–298), but a randomized control trial is needed to confirm these findings in the longer-term, particularly given the newly published data demonstrating long-term success with common obesity (299). More recently, the combination of tesofensine (a monoamine reuptake inhibitor) and metoprolol has shown promising results in a phase 2 trial (300).

Other hypothalamic symptoms include sleep-wake cycle disturbances, adipsia, temperature dysregulation, cognitive (particularly memory loss), and behavioral (particularly autistic) disorders. Children with disturbed sleep and/or behavioral difficulties should be referred to a specialist sleep laboratory and behavioral neuropsychopharmacology unit. These disorders are even more difficult to treat than replacement of the endocrine deficits. Where endocrine deficits, particularly ACTH deficiency and DI coexist, hypothalamic adipsia is potentially fatal particularly during intercurrent illness and surgery, requiring careful day-to-day fluid management with obligate daily fluid intake and desmopressin dose adjustments. The difficulties in managing patients with panhypopituitarism with concurrent hypothalamic dysfunction should not be underestimated, therefore avoiding these complications must be an important aim of initial therapy.

CONCLUSIONS

Pediatric hypothalamo-pituitary tumors are uncommon, and may present with occult or unusual clinical features posing diagnostic dilemmas that incur treatment delays or necessitate prolonged MRI surveillance. Notwithstanding their generally high survival rates, tumor- or treatment-related neuroendocrine morbidity is very significant and not always simply reversible by hormone replacement therapy. Consequently, treatment decision-making should aim to preserve not only visual, but also hypothalamo-pituitary function. Pediatric endocrinologists and pituitary surgeons should be part of the decision-making multidisciplinary team, with radiological, visual, and biochemical assessments together aiding management planning. A detailed baseline endocrine assessment is paramount to both diagnosis and treatment and will ultimately improve long-term outcome monitoring, the clarification of tumor- and treatment-related consequences and management of lifelong morbidity. Given the potentially significant reduction in health-related quality of survival, lifelong, age-appropriate follow-up and management within a dedicated multidisciplinary neuroendocrine unit familiar with the complexity of patients’ needs is recommended. To achieve this, rehabilitation, reproductive, neuropsychological, and vocational services need developing further in parallel with appropriate transition processes to adult services if we are to better manage and improve outcomes for this high-risk group of young patients. Evidence- and consensus-based guidelines are increasingly being developed to define a standard of best practice for the management of these rare tumors.

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Diabetes Insipidus

CLINICAL RECOGNITION

Diabetes Insipidus (DI) is the excess production of dilute urine. Diagnosis requires a targeted history, examination and confirmation through appropriate laboratory and radiological investigations. DI presents with polyuria and polydipsia. Urine output is more than 40 ml/kg /24 hours in adults and more than 100 ml/kg/24 hours in children. DI reflects either the lack of production or action of the posterior pituitary hormone vasopressin (AVP). There are three subtypes.

Cranial or hypothalamic DI (HDI): due to relative or absolute lack of AVP.
Nephrogenic DI (NDI): due to partial or total resistance to the renal antidiuretic effects of AVP.
Dipsogenic DI (DDI, primary polydipsia): where polyuria is secondary to excessive, inappropriate fluid intake.

All forms of DI are rare. HDI has an estimated prevalence of 1/25,000. While presentation is more common in adults, familial forms of both HDI and NDI characteristically present in childhood.

PATHOPHYSIOLOGY

The Physiology of AVP

AVP is a nine-amino acid peptide made within magnocellular neurones of the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus that project through the hypophyseal portal tract to terminate in the posterior pituitary, where AVP is released into the circulation. Together, the PVN, SON and posterior pituitary form an anatomical and functional unit- the neurohypophysis (Figure 1).

AVP is produced from a large precursor that undergoes extensive post-translational processing (figure 2).

The major action of AVP is in the regulation of renal water excretion. AVP increases expression of the AVP-dependent water channel Aquaporin 2, which is expressed in the renal collecting duct, facilitating water reabsorption. This action of AVP is mediated by the type 2 AVP receptor (V2-R), expressed exclusively on the interstitial surface of target cells in the distal nephron. AVP release is regulated by osmoreceptors within the lamina terminalis. There is a linear relationship between plasma osmolality and plasma AVP concentration. Not unexpectedly, thirst perception regulated in a parallel manner (Figure 3).

Hypothalamic DI

Presentation with HDI implies loss of 80%-90% of AVP production from the posterior pituitary. This, in turn, reflects either destruction of vasopressinergic magnocellular neurons in the hypothalamus or interruption of intra-axonal transport/processing of AVP. Some 50% of children and young adults with HDI have an underlying tumor or CNS malformation (e.g., craniopharyngioma, germinoma, septo-optic dysplasia). Familial HDI comprises 5% of cases.

Acute HDI can occur in up to 22% of non-selected patients presenting with traumatic brain injury (TBI), persisting in some 30% of these on long term follow up. HDI may follow trauma to the pituitary or hypothalamus. HDI following surgery to the pituitary or neurohypophysis presents within 24-48h after surgery and is often transient, resolving within 10 days. Pituitary stalk trauma (including that following surgery) may lead to a tri-phasic disturbance in water balance; an immediate polyuria due to HDI followed by a more prolonged period of antidiuresis suggestive of AVP excess. The antidiuretic phase may last several weeks and can be followed by reversion to HDI or recovery. DI presenting with a pituitary mass should raise concerns about a diagnosis other than pituitary adenoma. HDI can worsen in pregnancy due to increased degradation of AVP by placental enzyme activity

Table 1. Etiology of HDI
Primary
Genetic	Wolfram syndrome Autosomal dominant Autosomal recessive
Developmental syndromes	Septo-optic dysplasia
Idiopathic
Secondary/acquired
Trauma	Head injury, Post-surgery
Tumor	Craniopharyngioma Germinoma Metastases Pituitary macroadenoma
Inflammatory	Sarcoidosis, Histiocytosis, Meningitis, Encephalitis, Infundibuloneurohypophysitis, Guillain–Barré syndrome, Autoimmune

Nephrogenic DI

Renal resistance to AVP may reflect a toxic renal tubulopathy secondary to metabolic (e.g., hypokalemia; hypercalcemia) or drug effects (e.g., lithium). Prolonged polyuria of any cause can result in partial NDI through disruption of the intra-renal solute gradients and reduced tubular concentrating capacity.

X-linked familial NDI results from loss-of-function mutations in the renal AVP receptor (Figure 4). Autosomal recessive NDI is caused by loss-of-function mutations in the AVP-dependent renal water channel aquaporin-2.

Dipsogenic DI

Persistent high fluid intake leads to appropriate polyuria. If intake exceeds the limit of renal free water excretion,hyponatremia may result. DDI can be associated with abnormalities in thirst perception.

Low threshold for thirst
Exaggerated thirst response to osmotic challenge
Inability to suppress thirst at low plasma osmolalities

Neuroimaging is normal in most cases. DDI is associated with affective disorders.

DIAGNOSIS AND DIFFERENTIAL

History and examination may reveal important clinical information

-Features of systemic disease

-Associated endocrinopathy: suggestive of additional hypothalamic or pituitary dysfunction

-Neuro-ophthalmic problems suggestive of structural disease

-Evidence of drug toxicity (e.g., lithium, phenytoin)

There should be a standard initial diagnostic approach.

-Confirmation of true polyuria, distinct from simple frequency without excess urine volume

-Exclusion of common differentials such as drug (diuretics) and metabolic causes (hyperglycemia, hypercalcemia hypokalemia)

If polyuria is confirmed and simple causes are excluded, the clinician should proceed to a diagnostic Water Deprivation Test (Table 2)

Definitive diagnosis of DI requires testing of AVP production and action in response to osmolar stress. The water deprivation test is an indirect assessment of the AVP axis, measuring renal concentrating capacity in response to dehydration. It can be followed by assessment of renal response to the synthetic AVP analogue DDAVP, to determine whether any defect identified in urine concentrating ability can be corrected with AVP-replacement.

Table 2. Water Deprivation Test
Step 1 - Dehydration phase
Aim	Differentiate HDI and NDI from DDI
Procedure	Restrict all fluids between 8am-4pm in a controlled environment. Take baseline and 2 hourly measurements of weight, urine volume, urine osmolality, and plasma osmolality. Abandon test if thirst becomes unbearable or if patient loses >5% initial weight.
Analysis	HDI and NDI: Urine osmolality <300mOsm/kg Plasma osmolality >290mOsm/kg DDI: Urine and plasma osmolality normal
Step 2 – DDAVP (desmopressin) response phase
Aim	Differentiate HDI from NDI
Procedure	At 4pm, administer desmopressin bolus (1mcg, intramuscular). Allow fluid intake up to 2x the volume of urine output in step 1. Continue to measure urine volume, urine osmolality and plasma osmolality every hour until 8pm. Measure plasma osmolality and plasma sodium at 9am the next morning.
Interpretation	HDI: Urine osmolality >750 mOsm/kg NDI: Urine osmolality remains low

Further Investigations

Water deprivation test results may be indeterminate. If HDI is suspected but water deprivation test data are inconclusive, a reasonable approach is a therapeutic trial of 10-20 mcg intranasal DDAVP per day with close monitoring of plasma Na⁺. Patients with HDI note improved symptoms without significant dilutional hyponatremia. In the future, basal or stimulated measurement of copeptin may be the most useful investigation, when generally available.

Confirmation of HDI should lead to further pituitary function testing and cranial MRI. MRI may reveal the absence of posterior pituitary bright spot on T1- weighted sequences (Figure 5), or a pituitary mass. In the absence of structural problem, the MRI should be repeated 12 months after presentation to exclude slow growing mass lesion. NDI requires renal tract imaging and additional renal studies.

Diabetes Insipidus Combined with Defects in Thirst (Adipsic DI)

While the regulation of thirst and AVP are discrete, the close neuroanatomical relationship of the structures responsible for osmoregulation of both processes means that some structural, neurovascular and neuro-developmental lesions are associated with combined defects. Absent or reduced thirst (adipsia) in association with HDI predisposes to hypernatremic dehydration. Diagnosis follows that outlined for HDI, with parallel assessment of thirst perception.

TREATMENT

Mild forms of HDI may not require treatment. Significant polyuria and polydipsia are treated effectively with DDAVP in divide doses: nasal spray 5-100 mcg per day; tablets 100-1000 mcg/day; or parenterally 0.1-2.0 mcg/day. Hyponatremia from plasma dilution can be avoided by omitting treatment for a short period on a regular basis (e.g., one dose per week).

NDI may respond to removal of the causal agent (such as correction of hypokalemia or cessation of Lithium). However, drug-induced NDI may persist. Symptoms may respond partly to high-dose DDAVP (e.g., 4 mcg i.m. bid.) Hydrochlorothiazide (25 mg/day) either alone or in combination with Ibuprofen (200 mg/day) may be of some help. Urine output should not be expected to normalize.

The approach to DDI is reduction in fluid intake. DDAVP treatment must be avoided because of the risk of significant hyponatremia.

Patients with adipsic DI require careful management. Absence of normal thirst perception and/or regulation means that they may continue to drink at low plasma osmolalities that would normally suppress fluid intake. The combination of an obligate antidiuresis produced by DDAVP treatment, together with the potential for spontaneous fluid intake in excess of that required for maintenance of plasma volume and normal plasma osmolality, means they are at risk of fluid overload and dilutional hyponatremia. The same group of patients are also at risk of dehydration and hypernatremia if total body water loss is higher than a spontaneous fluid intake that is, by definition, uncoupled from normal osmo-regulatory control. In patients with adipsic DI, managing fluid balance to maintain normal plasma sodium is therefore challenging. One approach is to combine a fixed DDAVP-dependent antidiuresis (giving urine output of some 2 L/day) with a variable daily fluid intake that aims to maintain the patient’s body weight at that which is known to be associated with normal plasma volume and normal plasma sodium (the ‘target’ weight, see below).

e.g. Fluid intake for given day (L) = 2 L (i.e., urine output from fixed dose DDAVP) - (weight on given day in kg - target weight in kg)

FOLLOW-UP

Following initiation of DDAVP, patients require review for dose titration. When stable, they can be seen annually to assess symptom control and to check plasma Na⁺ levels to avoid over-treatment. Adipsic DI requires meticulous follow-up in a specialist service.

GUIDELINES

Baldeweg SE, Ball S, Brooke A, Gleeson HK, Levy MJ, Prentice M, Wass J. In-patient management of Cranial Diabetes Insipidus. Endocrine Connections 2018;

REFERENCES

Ball SG. The Neurohypophysis: Endocrinology of Vasopressin and Oxytocin. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2017 Apr 22. PMID: 25905380

Gubbi S, Hannah-Shmouni F, Koch CA, Verbalis JG. Diagnostic Testing for Diabetes Insipidus. 2019 Feb 10. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, Dungan K, Hershman JM, Hofland J, Kalra S, Kaltsas G, Koch C, Kopp P, Korbonits M, Kovacs CS, Kuohung W, Laferrère B, Levy M, McGee EA, McLachlan R, Morley JE, New M, Purnell J, Sahay R, Singer F, Sperling MA, Stratakis CA, Trence DL, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000–. PMID: 30779536

Ball S 2013. Diabetes Insipidus. Medicine 41: 519-521. 10.1016/j.mpmed.2013.06.001

http://www.sciencedirect.com/science/article/pii/S1357303913001783

Refardt J, Winzeler B, Christ-Crain M. Endocrinol Metab Clin North Am. 2020 Sep;49(3):517-531. doi: 10.1016/j.ecl.2020.05.012. Epub 2020 Jul 15. PMID: 32741486

Garrahy A, Thompson CJ. Management of central diabetes insipidus. Best Pract Res Clin Endocrinol Metab. 2020 Sep;34(5):101385. doi: 10.1016/j.beem.2020.101385. Epub 2020 Jan 31. PMID: 32169331

Hypopituitarism: Emergencies

CLINICAL RECOGNITION

Hypopituitarism usually has an insidious presentation over weeks to months and often remains clinically silent until a particular stressful event such as a concurrent infection or trauma at which time marked symptoms are evident. The reason for a protracted course until deficiency is clinically evident is due to the slow depletion of pituitary hormones. The end endocrine organs also have other, albeit less effective, ways of dealing with lack of pituitary input (for example non-ACTH stimulation of cortisol from the adrenal or constitutive activation of the TSH receptor at a low level in the thyroid). The presentation of hypopituitarism is different from the catastrophic clinical situation such as a hemorrhagic infarct into the pituitary that results in acute pituitary insufficiency and cardiovascular collapse. The latter is referred to as pituitary apoplexy and is discussed in another chapter. The more common presentation is in a patient who slowly develops fatigue and rather nonspecific symptoms.

Each cell in the pituitary is responsible for one or more pituitary hormones and hyposecretion of the hormone can result in a variety of symptoms as summarized in Table 1. Although the symptoms are rarely pathognomonic for hypopituitarism or a particular hormone insufficiency, collectively the presence of symptoms such as body fatigue and failure to thrive with or without cardiovascular compromise in the right clinical scenario, should alert the physician to a pituitary etiology.

Table 1. Signs of Symptoms of Hypopituitarism in Adults
Cell Type, Pituitary Hormone	Affected Hormone	Symptoms/Signs
Corticotrophs, ACTH	Cortisol	Hypoglycemia
		Hyponatremia
		Hypotension -> Shock
Thyrotrophs, TSH	T4, T3	Confusion -> Coma
		Hypothermia
		Bradycardia
		Hyponatremia
		Lethargy
		Edema
Gonadotrophs, LH/FSH	Testosterone/Estrogen	Deceased muscle mass
		Deceased libido
		Deceased muscle strength
		Hair loss
		Amenorrhea
		Infertility
Somatotrophs, GH	GH/IGF-1	Decreased muscle mass
		Lethargy
Lactotrophs, Prolactin		Failure of lactation

Often times, patients with panhypopituitarism can present with slight elevation, as opposed to deficiency, in prolactin levels causing amenorrhea and/or galactorrhea. This is because damage to the pituitary stalk (usually from mass effect) can cause interruption of the continuous dopaminergic inhibition of the lactotrophs resulting in elevated prolactin levels (usually less than 200 ng/mL). It is important to be able to differentiate this entity from a prolactinoma, which usually presents with levels above 200ng/mL.

Hypopituitarism can also manifest with deficiency of vasopressin (AVP) if there is damage to the posterior pituitary. This will cause the clinical syndrome known as diabetes insipidus, which classically manifests with polyuria, polydipsia, hypernatremia, and low urine osmolarity.

Often it will be difficult for the physician at the bedside to determine whether the suspected hormone deficiency is in fact due to a pituitary insufficiency or primary failure of one of the major endocrine glands such as the adrenal, thyroid, or gonads. While some clues may come from the history, the suggestion that one or more endocrine glands are dysfunctional should alert the physician to a pituitary etiology. The presenting signs and symptoms are similar in both children and adults although the presentation in children is usually much more dramatic. The signs are more often associated with cardiovascular instability, failure to gain weight or grow depending on the degree of pituitary hormone deficiency and can be present at birth or later.

PATHOPHYSIOLOGY

The etiologies of hypopituitarism are either congenital or acquired. While congenital hypopituitarism is usually associated with early onset hemodynamic instability, growth disturbances and failure to thrive the symptoms may not manifest until puberty when a surge in pituitary hormones is required for normal physiology, and puberty is halted. Although isolated pituitary hormone deficiencies are found (usually due to genetic defects in specific pituitary cell transcription factors), when two pituitary cell lines are impaired it is generally an indication that all five cell lines are malfunctioning. There is however, a predictive order of loss of hormonal function, with a tendency to preserve the most crucial hormones for survival. (Usually manifesting first with loss of somatotrophs and gonadotrophs and last with loss of corticotroph function). The acquired causes of hypopituitarism are listed in Table 2. One should also consider the possibility of hypothalamic disease as a cause of pituitary insufficiency.

Table 2. Causes of Hypopituitarism

Congenital

Gland Malformation

Transcription Factor Defects

Acquired

Destruction due to tumors (e.g. Craniopharyngioma, non-secreting pituitary adenomas, hamartomas)

Infection (e.g. Tuberculosis)

Destruction due to inflammation (e.g. Sarcoid, Hemochromatosis)

Postsurgical

Post-radiation

Hemorrhage

Abrupt Hormone Therapy Withdrawal

DIAGNOSIS AND DIFFERENTIAL

Diagnostic Tests

ACTH

There is a general lack of enthusiasm in measurement of static hormones for the diagnosis of hypopituitarism This is in part due to the variable nature of hormone secretion in normal physiological states. Cortisol is released from the adrenal gland in a pulsatile fashion under the direction of ACTH. Furthermore, ACTH secretion is responsive to the hypothalamic factor, corticotropin releasing hormone (CRH), which is also released in an episodic manner. Therefore, depending on the instance that blood is sampled; there can be significant variation in the absolute values of ACTH and cortisol.

The hypothalamic factor, CRH is not readily measured in the blood and the normal reference values have not been established in the literature.

It is important to keep in mind that measurement of total cortisol in serum is also influenced by the presence of cortisol binding globulin (CBG) which can be affected by clinical scenarios like liver failure, sepsis, and high estrogen states, such as pregnancy and use of oral contraceptives.

Provocative tests are more useful in the assessment of the hypothalamic-pituitary-adrenal axis than are static and unstimulated values of hormones. The screening test of choice to rule out adrenal insufficiency (both primary and secondary) is the 8:00 serum cortisol level (ruled out if >20 µg/dL but can vary depending on the assay). The ACTH stimulation test can be used for confirmation (Table 3) for summary of tests). This test however, might not be able to diagnose acute secondary adrenal insufficiency in which case, it might be necessary to perform the insulin tolerance or metyrapone stimulation test.

TSH

Unlike cortisol levels, static thyroid hormone levels can provide valuable diagnostic information. While there is also pulsatile fluctuation in serum concentrations of TSH, the excursions are much less than with cortisol due to the longer half-life of T4 (7 days versus minutes for cortisol).

It is important to point out however, that the clinician should never rely on the measurement of an isolated TSH level (without free T4 measurement) when suspecting secondary hypothyroidism, as this entity can often present itself with an inappropriately normal TSH level in the presence of a low T4.

GONADOTROPINS

Similar, to the thyroid, gonadal hormones (testosterone and estrogen) are readily measured in the blood and are more stable and less disturbed by pulsatile secretion. Baseline AM measurement of the gonadal hormone LH and FSH can be useful to distinguish primary (gonadal) versus secondary (pituitary) disease. It is worth mentioning that although unlikely to cause changes in clinical management, a low FSH level in a postmenopausal woman can be a very sensitive test to screen for hypopituitarism when clinically suspected.

Table 3. Tests Used in the Diagnosis of Hypopituitarism
Test for Hormonal Deficiency	Expected Result if Deficient
ACTH
8:00 AM Cortisol	<20 µg/dl
Insulin Tolerance (0.1U/Kg)	Cortisol <20µg/dl
ACTH Stimulation (250 µg)	Cortisol <20 µg/dl*
Metyrapone stimulation test	ACTH <75pg/mL
TSH
T4/T3/TSH	Dec./Dec./Dec or not elevated
Gonadotropins
Testosterone or Estradiol	Lower than reference range
LH/FSH	Normal or lower than reference range
Growth Hormone
IGF-1	Lower than reference range
Insulin Tolerance (0.1U/Kg)	Growth Hormone <5.1 ng/ml
Glucagon Stimulation (1mg)	<3 ng/ml
*May not be abnormal in acute hypopituitarism as adrenal response to ACTH may remain intact. Note- hormone levels that are considered abnormal will vary depending upon the assay used

Imaging Studies

Imaging studies, namely a dedicated MRI of the pituitary, are important in determining the presence of a structural lesion, however the presence (or absence) of a tumor or mass does not always correlate with pituitary function.

TREATMENT

The objective of treatment of hypopituitarism is to replace deficient hormones. It is usually not practical to directly replace the pituitary hormone, but rather treatment is with the end-organ hormone (e.g. thyroid hormone is used for TSH deficiency rather than TSH and corticosteroids for ACTH deficiency rather than ACTH).

In general, it is recommended to begin by replacing the hormones with more critical metabolic functions first. Glucocorticoids should be instituted first to avoid an adrenal crisis, followed by thyroid replacement therapy and after this if appropriate, gonadal and growth hormone replacement.

Titration of glucocorticoid replacement is quite challenging, as one cannot rely on cortisol or ACTH levels to assess for under or over replacement. The corticosteroid replacement dose is usually estimated based on body mass weight and delivered at different doses throughout the day trying to mimic as much as possible its physiologic circadian rhythm. The recommended doses in table 4 are guidelines only and need to be titrated by the bedside physician based on the specific clinical situation. It is extremely important to emphasize to other clinicians and patients with adrenal insufficiency, that during acute sickness and high stress situations, higher doses of glucocorticoid replacement are needed.

Thyroid replacement therapy is somewhat easier to titrate, as free T4 levels can be quite useful. The clinician however, should avoid the mistake of following TSH levels, as they will not be useful in secondary hypothyroidism.

Table 4. Treatment of Hypopituitarism
Pituitary Hormone/ Treatment	Acute	Chronic Deficiency
ACTH
Hydrocortisone	50-100 mg IV Q8H
Hydrocortisone		15 mg q AM, 5 mg q3PM
TSH
Levothyroxine	1.6 ug/kg daily	1.6 ug/kg daily
LH/FSH
Testosterone (men)		Transdermal-5 gm qD
		IM – Test. Cypionate 200 mg q 2 weeks
Estrogen (women)		Varies
Growth Hormone
Growth Hormone	No acute indication	0.05 mg/kg/d

For details of hormone therapy see the appropriate Endotext chapters

FOLLOW-UP

After the diagnosis is made and acute treatment is started (Table 4, Acute) a decision needs to be made whether continued chronic therapy with hormone replacement is needed. A month after discharge from the hospital and recovery from the acute event, if necessary, patients are retested to determine if the endocrine defect persists. This will depend on the etiology as removal of tumor or reversal of an infiltrative process sometimes allows recovery of function. Repeat testing will confirm whether the patient needs to remain on life-long hormone replacement therapy.

GUIDELINES

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

REFERENCES

Yeo KT, Babic N, Hannoush Z, Weiss RE. Endocrine Testing Protocols: Hypothalamic Pituitary Adrenal Axis. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2017 May 17.

Chung TT, Koch CA, Monson JP. Hypopituitarism. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2018 Jul 25.

Pituitary Apoplexy

CLINICAL RECOGNITION

Insult to the pituitary can be in the form of hemorrhage, infarction, or both. When abrupt and sometimes catastrophic hemorrhagic infarction occurs in the pituitary it is defined as apoplexy. The constellation of headache, vomiting, visual impairment (Apoplexy Triad), and altered consciousness with hemodynamic instability, although not specific for pituitary apoplexy, are reasons to consider the diagnosis (Table 1). Often the presentation in this dramatic fashion is the first time the patient is aware that he/she harbors a pituitary tumor. Asymptomatic hemorrhage and infarct into a pituitary tumor can occur in 10-25% of patients, however true apoplexy (the constellation of symptoms noted above) occurs in 2-10% of pituitary tumor patients.

Table 1. Signs and Symptoms of Pituitary Apoplexy
Symptom	Incidence
Headache	95%
Vomiting	70%
Vision Defects: Visual field defect Decreased visual acuity Diplopia (CN III, IV, V and VI)	64% 52% 78%
Hemiplegia	Rare
Meningismus	Rare
Hypotension (cardiovascular collapse)	95%

The most common presenting complaint, headache, can present variably from retroorbital to unilateral to bilateral temporal headaches. These are the symptoms during the acute phase of apoplexy. As the hemorrhagic infarct resolves often times the patient is left with hypopituitarism (refer to section on Hypopituitarism).

Certain conditions will predispose a patient to the catastrophe of pituitary apoplexy. (Table 2). While all large pituitary tumors are at risk for hemorrhagic infarction, certain functional pituitary tumors such as those in Cushing’s disease or acromegaly may be particularly prone. Nearly 25% of all patients with apoplexy have inadequately treated hypertension.

Table 2. Predisposing Conditions Associated with Pituitary Apoplexy

Pituitary Tumor

Non-functioning pituitary macroadenoma

Certain functional tumors

Hypertension and/or hypotension

Surgery

Cardiac surgery (heart lung bypass; coronary artery grafts)

Major orthopedic procedures

Drugs

Endocrine stimulation tests (Thyrotropin releasing hormone stimulation; insulin tolerance test)

Anticoagulants

Estrogen

Head Trauma

Pregnancy and Delivery

Infections

Dengue fever

Hypophyisitis

Radiation therapy

PATHOPHYSIOLOGY

It is thought that alterations in blood flow to pituitary adenomas coupled with high metabolic demands lead to apoplexy. The main symptoms and consequences of apoplexy are due to the increased pressure present within the bony walls of the sella turcica in which the pituitary resides. A sudden increase in the sella contents due to blood and edema results in increased pressure. This increased pressure and meningeal irritation are responsible for the neurologic symptoms described in Table 1, including the increased pressure in the cavernous sinus and the cranial nerve palsies as well as bitemporal-hemianopsia. Extravasation of blood into the subarachnoid space causes meningeal irritation.

DIAGNOSIS AND DIFFERENTIAL

Physicians must promptly recognize that patients presenting with the triad of headache, vomiting, and visual disturbances may have any one of several diagnoses that require urgent attention to prevent death or irreversible neurologic impairment.

Clinical Evaluation

Evaluation of the patient should begin with a thorough history, from the patient if sufficiently conscious to give one, or from family members. A history of a pituitary tumor should raise the suspicion for apoplexy. More subtle abnormalities associated with pituitary dysfunction (hypothyroidism, adrenal insufficiency, or hypogonadism) may be helpful (see section on Hypopituitarism).

Radiologic and Laboratory Evaluation (Table 3)

The cornerstone for diagnosis of patients presenting with the Apoplexy Triad is urgent radiologic assessment. MRI T2 weighted images are the test of choice and should be performed emergently in all patients with visual symptoms. A CT scan can be useful when an MRI is neither available or possible.

Urgent measurement of blood chemistries, including electrolytes, kidney function, liver function, complete blood count with platelets, and prothrombin time can be useful. Since more than 80% of patients will have endocrine dysfunction, urgent measurement of free T4, TSH, prolactin, ACTH, and a random cortisol can be helpful and usually is readily available. Less rapidly available and helpful (and less important in the initial diagnosis and management) are other pituitary hormones such as LH, FSH, estradiol or testosterone, growth hormone, and IGF I.

Examination of cerebral spinal fluid is usually not diagnostic, and unnecessary if the diagnosis of apoplexy is certain. However, if there is bleeding into the CSF as a result of the apoplexy, red blood cells as well as elevated protein and xanthochromia can be seen.

Table 3. Useful Tests in the Diagnosis of Pituitary Apoplexy
TEST	Expected Result in Apoplexy
MRI pituitary	Hemorrhagic infarct in region of pituitary
Electrolytes	Hyponatremia,
Complete Blood Count	Anemia, thrombocytopenia
Prothrombin time	Possibly prolonged
FT4/TSH	Low/Low or normal
Prolactin	Low (< 1 ng/dl)
Cortisol, random	Usually < 5 ug/dl
Other tests of the endocrine axes	See section on Hypopituitarism
Visual Field Testing	Defect

The differential diagnosis of pituitary apoplexy should include other conditions that result in the symptoms of headache, vomiting, visual disturbances, and hemodynamic instability (Table 4). Each of these conditions is itself a medical emergency that requires specific treatment.

Table 4. Differential Diagnosis of Pituitary Apoplexy

Subarachnoid hemorrhage (can be distinguished from apoplexy by MRI with MR angiogram)

Infectious meningitis

Cavernous sinus thrombosis

Migraine

Rathke cyst hemorrhage

Hyperemesis gravidarum

Stroke

TREATMENT

The key to successful management of patients with pituitary apoplexy is a team approach including critical care neurologists, neurosurgeons, neuro-ophthalmologists, and endocrinologists. Together each of these specialists provide needed expertise in the management and ongoing care of these patients. Acute secondary adrenal insufficiency is seen in approximately two-thirds of patients and is the major source of mortality associated with this condition. Prompt glucocorticoid replacement is there for mandatory and should not be delayed for confirmatory testing. The initial management is stabilization of the hemodynamic status with IV 0.9% NaCl boluses to maintain normal tissue perfusion, and usually high dose parenteral glucocorticoids (100 mg hydrocortisone q 8h intravenous). Unless significant cerebral edema is present, hydrocortisone rather than dexamethasone is favored.

Although there is a general consensus that patients with pituitary apoplexy and significant neuro-ophthalmic signs or reduced level of consciousness should have surgical decompression, there is significant controversy in the best timing for the surgical procedure due to lack of good quality outcomes data. (see figure 1 for a suggested management approach).

Figure 1. Treatment of Pituitary Apoplexy.

FOLLOWUP

While 80% of patients have residual hypopituitarism following apoplexy (with or without surgical decompression) some patients do not display immediate evidence of hypopituitarism. In addition, recurrent apoplexy and tumor regrowth has been reported to occur. MRI of the pituitary should be obtained at 3–6-month intervals until the anatomy is stable and then yearly for 5 years. A month after discharge from the hospital and recovery from the acute event, if necessary, patients are subject to repeat endocrine testing to determine if the endocrine defects persist. Repeat testing will confirm whether the patient needs to remain on life-long hormone replacement therapy.

GUIDELINE

Rajasekaran, S., Vanderpump, M., Baldeweg, S., Drake, W., Reddy, N., Lanyon, M., Markey A., Plant, G., Powell, M., Sinha, S., Wass, J. UK guidelines for the management of pituitary apoplexy. Clin Endocrinol (Oxf) 2011 Jan 74(1);9-20. PMID 21044119 http://www.ncbi.nlm.nih.gov/pubmed/21044119

REFERENCES

Chung TT, Koch CA, Monson JP. Hypopituitarism. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2018 Jul 25
Vanderpump M1, Higgens C, Wass JA. UK guidelines for the management of pituitary apoplexy a rare but potentially fatal medical emergency. Emerg Med J. 2011 Jul;28(7):550-1.
Donegan D, Erickson D. Revisiting Pituitary Apoplexy. J Endocr Soc. 2022 Jul 26;6(9):bvac113. doi: 10.1210/jendso/bvac113. eCollection 2022 Sep 1. PMID: 35928242

ABSTRACT

INTRODUCTION

DEFINITION AND EPIDEMIOLOGY

ENERGY HOMEOSTASIS

MOLECULAR GENETICS OF BODY FATNESS

Heritability of Body Fatness

Common Single Gene Mutations Associated with Obesity

Genome-Wide Association Studies (GWAS) of the Obesity Phenotype

ENVIRONMENTAL FACTORS AFFECTING PEDIATRIC RISK OF OBESITY AND ADIPOSITY-RELATED MORBIDITIES

Epigenetics

Early Feeding Practices

Early Life BMI Trajectories

Social Determinants of Health

Physical Activity, Sedentary Behavior, and Sleep

MORBIDITIES ASSOCIATED WITH OBESITY IN CHILDREN

Pediatric Obesity and Cardiovascular Risk Factors

Pediatric Obesity and Type 2 Diabetes Mellitus

ENDOCRINE CHANGES ASSOCIATED WITH OBESITY IN CHILDREN

WEIGHT STIGMATIZATION (FAT SHAMING)

PREVENTION AND TREATMENT OF OBESITY: CLINICAL APPROACH TO THE PEDIATRIC PATIENT

Prenatal Care

Initial Evaluation

Therapeutic Intervention

DIETARY RECOMMENDATIONS

THERAPEUTIC EXERCISE

DIGITAL INTERVENTIONS

PHARMACOLOGICAL AND SURGICAL INTERVENTIONS

CONTRINDICATIONS TO BARIATRIC SURGERY INCLUDE:

ADDRESSING WEIGHT STIGMATIZATION

OTHER INTERVENTIONS

REFERENCES

ABSTRACT

INTRODUCTION

EFFICACY OF HIGH-INTENSITY, IN-PERSON LIFESTYLE INTERVENTION PROGRAMS

Diabetes Prevention Program

Look AHEAD (Action for Health in Diabetes) Study

LIFESTYLE INTERVENTION COMPONENTS

Dietary Recommendations

Physical Activity Recommendations

PHYSICAL ACTIVITY AND WEIGHT LOSS

PHYSICAL ACTIVITY AND WEIGHT MAINTENANCE

PHYSICAL ACTIVVITY AND CARDIOVASCULAR HEALTH

Principles of Behavior Therapy

GOAL SETTING

SELF-MONITORING

STIMULUS CONTROL

COGNITIVE STRATEGIES

STRUCTURE OF IN-PERSON BEHAVIORAL TREATMENT: SHORT- AND LONG-TERM

REMOTELY-DELIVERED LIFESTYLE MODIFICATION INTERVENTIONS

Telehealth Delivery

Digital Delivery via the Internet or Smartphone

CONCLUSION

REFERENCES

ABSTRACT

INTRODUCTION

WHEN TO CONSIDER ISLET TRANSPLANTATION?

MULTIDISCIPLINARY TEAM

ISLET ISOLATION AND TRANSPLANTATION

THE CONSORTIUM CONCEPT

ISLET TRANSPLANT ACTIVITY

The Collaborative Islet Transplant Registry (CITR)

Outside The Collaborative Islet Transplant Registry (CITR)

Clinical Management of Islet Transplant Recipients

Immunosuppression

Antibiotic and Antiviral Prophylaxis

Thromboembolism Prophylaxis

Peri-transplant Insulin Management

POST-TRANSPLANT CLINICAL MONITORING

MONITORING ISLET GRAFT FUNCTION

The Igls Score

IMPACT OF ISLET TRANSPLANTATION ON METABOLIC CONTROL (TABLE 6)

IMPACT OF ISLET TRANSPLANTATION ON DIABETES COMPLICATIONS

COMMON ADVERSE EVENTS AND THEIR MANAGEMENT (TABLE 7)

CURRENT CHALLENGES

FUTURE DEVELOPMENTS IN BETA-CELL REPLACEMENT THERAPIES

CONCLUSIONS

ACKNOWLEDGMENTS

ONLINE RESOURCES ON THE SUBJECT

ADDITIONAL ONLINE RESOURCES IN RELATED TOPICS

REFERENCES