Thyroid Hormone Synthesis and Secretion

ABSTRACT

The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the regulation of metabolic processes throughout the body. As at any factory, effective production depends on three key components - adequate raw material, efficient machinery, and appropriate controls. Iodine is the critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where it reduces H₂O₂, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to tyrosyls in Tg. H₂O₂ is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about 70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available for recycling within the gland. TSH is the stimulator that affects virtually every stage of thyroid hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply, either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can produce goiter and hypothyroidism. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.

INTRODUCTION

The thyroid contains two hormones, L-thyroxine (tetraiodothyronine, T₄) and L-triiodothyronine (T₃) (Figure 2-1, below). Iodine is an indispensable component of the thyroid hormones, comprising 65% of T₄'s weight, and 58% of T₃'s. The thyroid hormones are the only iodine-containing compounds with established physiologic significance in vertebrates.

Fig. 2-1: Structural formula of thyroid hormones and precursor compounds

The term "iodine" occasionally causes confusion because it may refer to the iodine atom itself but also to molecular iodine (I₂). In this chapter "iodine" refers to the element in general, and "molecular iodine" refers to I₂. "Iodide" refers specifically to the ion I^-.

Ingested iodine is absorbed through the small intestine and transported in the plasma to the thyroid, where it is concentrated, oxidized, and then incorporated into thyroglobulin (Tg) to form MIT and DIT and later T₄ and T₃ (Figure 2-2). After a variable period of storage in thyroid follicles, Tg is subjected to proteolysis and the released hormones are secreted into the circulation, where specific binding proteins carry them to target tissues. This chapter discusses these broad steps as: (a) iodine availability and absorption; (b) uptake of iodide by the thyroid; (c) oxidation of iodide, which involves the thyroperoxidase (TPO), H₂O₂, and H₂O₂ generation; (d) Tg, whose iodination leads to hormone formation; (e) storage of thyroid hormones in a Tg-bound form; (f) Tg breakdown and hormone release; (g) control of synthesis and secretion by iodine supply and TSH; and (h) effects of drugs and other external agents on the process.

Fig. 2-2: The iodide cycle. Ingested iodide is trapped in the thyroid, oxidized, and bound to tyrosine to form iodotyrosines in thyroglobulin (TG); coupling of iodotyrosyl residues forms T₄ and T₃. Hormone secreted by the gland is transported in serum. Some T₄ is deiodinated to T₃. The hormone exerts its metabolic effect on the cell and is ultimately deiodinated; the iodide is reused or excreted in the kidney. A second cycle goes on inside the thyroid gland, with deiodination of iodotyrosines generating iodide, some of which is reused without leaving the thyroid.

The production of thyroid hormones is based on the organization of thyroid epithelial cells in functional units, the thyroid follicles. A single layer of polarized cells (Fig. 2-4A) forms the enveloppe of a spherical structure with an internal compartment, the follicle lumen. Thyroid hormone synthesis is dependent on the cell polarity that conditions the targeting of specific membrane protein, either on the external side of the follicle (facing the blood capillaries) or on the internal side (at the cell-lumen boundary) and on the tightness of the follicle lumen that allows the gathering of substrates and the storage of products of the reactions.Thyroid hormone secretion relies on the existence of stores of pre-synthetized hormones in the follicle lumen and cell polarity-dependent transport and handling processes leading to the delivery of hormones into the blood stream.

IODINE AVAILABILITY AND TRANSPORT

The daily iodine intake of adult humans varies from less than 10 µg in areas of extreme deficiency to several hundred milligrams for some persons receiving medicinal iodine. Milk, meat, vitamin preparations, medicines, radiocontrast material, and skin antiseptics are important sources (Table 2-1) (1;2). In the United States, the average intake in 1960 was about 100-150 µg/day, then rose to 200-500 µg/day in the following decade. It is currently about 150 µg/day (3). The use of iodate as a bread conditioner in the baking industry greatly increased average iodine consumption; this additive has been replaced more recently by other conditioners that do not contain iodine. Iodophors as sterilizing agents in the milk industry also added much iodine to the food chain, but this source may also be diminishing. In the USA and elsewhere, most consumers are unaware of the amount of iodine they ingest. Commerce and manufacturing technology rather than health dictate the presence of iodine in most products. The amounts of iodine are usually unrevealed, and changes in them unannounced.
In the USA, where iodized salt use is optional, about 70% of the population consumes table salt containing approximately 76 ppm iodine (76 mg I/kg salt). Most prepared food in the USA and Europe uses uniodized salt (Switzerland and Macedonia are exceptions) and only about 15% of the daily salt intake is added at the table, so iodized salt in these areas makes only a modest contribution to daily iodine intake (4). The National Health and Nutrition Examination Surveys (NHANES) showed that the median national urinary iodine excretion in the USA in samples collected between 1988 and 1994 was 145 µg/L, a marked decrease from the 321 µg/L in a similar survey two decades before (5;6). Estimates from the NHANES (2001) are about 160 µg/L. The Total Diet Study of the U.S. Food and Drug Administration reported a parallel decrease in iodine consumption between 1970 and 1990 (7) . These fluctuations in iodine intake result from changes in societal and commercial practices that are largely unrecognized and unregulated. Canada mandates that all salt for human consumption contain KI at 100 ppm (76 ppm as iodine). Calculations of the representative Canadian diet in 1986 estimated slightly over 1 mg iodine/person/day, of which iodized salt contributed over half (8). Urinary iodine excretion in a group of men in Ottawa in 1990 was less than 50% of that in the Canadian national survey of 1975 (9), suggesting a decrease in dietary intake there as well as in the USA. Some countries have areas with very high iodine intake (10), from dietary custom (e.g., seaweeds in Japan) or from iodine-rich soil and water (e.g., a few places in China). But many countries have had some degree of iodine deficiency (11) in at least part of their territory. This has been corrected by the widespread programs of iodine prophylaxis promoted by ICCIDD (12).
Too much iodine increases the incidence of iodine-induced hyperthyroidism, autoimmune thyroid disease and perhaps thyroid cancer. Too little causes goiter, hypothyroidism and their consequences i.e.features of the so-called iodine deficiency disorders (5). The global push to eliminate iodine deficiency in the current decades has put both excess and deficiency of iodine in the spotlight. Some countries have already moved rapidly from severe iodine deficiency to iodine excess, while others are only now recognizing iodine deficiency as a problem (5;12). Their experience, as well as that in the USA and Canada, emphasizes the need for continued monitoring to assess trends in iodine intake.
Medicinal sources can provide iodine in amounts much larger than those consumed in an average diet (Table 2-1). For example, 200 mg of amiodarone contains 75 mg of iodine. Radiographic contrast materials typically contain grams of iodine in covalent linkage, and significant amounts (milligrams) may be liberated in the body. Skin disinfectants (e.g., povidone iodine) and iodine-based water purification systems can greatly augment iodine intake. At the other end, some individuals with little consumption of dairy products and of iodized salt have low iodine intakes.

Table 2-1. Some common sources of iodine in adults USA (1,2)
Dietary iodine	Daily intake (µg)
Dairy products	52
Grains	78
Meat	31
Mixed dishes	26
Vegetables	20
Desserts	20
Eggs	10
Iodized salt	380
Other iodine sources	(µg)
Vitamin/mineral prep (per tablet)	150
Amiodarone (per tablet)	75,000
Povidone iodine (per mL)	10,000
Ipodate (per capsule)	308,000

Most dietary iodine is reduced to iodide before absorption throughout the gut, principally in the small intestine. Absorption is virtually complete. Iodinated amino acids, including T₄ and T₃, are transported intact across the intestinal wall. Short-chain iodopeptides may also be absorbed without cleavage of peptide bonds (13). Iodinated dyes used in radiography are absorbed intact, but some deiodination occurs later. Except in the postabsorptive state, the concentration of iodide in the plasma is usually less than 10 µg/L. Absorbed iodide has a volume of distribution numerically equal to about 38% of body weight (in kilograms) (14), mostly extracellular, but small amounts are found in red cells and bones.

The thyroid and kidneys remove most iodide from the plasma. The renal clearance of iodide is 30-50 mL plasma/min (14-16) and appears largely independent of the load of iodide or other anions. In certain species, such as the rat, large chloride loads can depress iodide clearance. In humans, renal iodide clearance depends principally on glomerular filtration, without evidence of tubular secretion or of active transport with a transfer maximum (17). Reabsorption is partial, passive, and depressed by an extreme osmotic diuresis. Hypothyroidism may decrease and hyperthyroidism may increase renal iodide clearance, but the changes are not marked (14;18).

On iodine diets of about 150 µg/day, the thyroid clears iodide from 10-25 mL of serum (average, 17 mL) per minute (14). The total effective clearance rate in humans is thus 45-60 mL/min, corresponding to a decrease in plasma iodide of about 12%/hr. Thyroidal iodide clearance may reach over 100 mL/min in iodine deficiency, or as low as 3 or 4 mL/min after chronic iodine ingestion of 500-600 µg/day.

The salivary glands and the stomach also clear iodide and small but detectable amounts appear in sweat and in expired air. Breast milk contains large amounts of iodide, mainly during the first 24 hours after ingestion (19). Its content is directly proportional to dietary iodine. For example, in one part of the USA with community adult iodine intake of about 300 µg daily per person, breast milk contained about 18 µg iodine/dL, while in an area of Germany consuming 15 µg iodine per capita daily, the breast milk iodine concentration was only 1.2 µg/dL (20). Milk is the source of virtually all the newborn's iodine, so milk substitutes need to provide adequate amounts.

UPTAKE OF IODINE BY THE THYROID

Thyroid cells extract and concentrate iodide from plasma (21;22). As shown in Fig. 2-3, shortly after administration, radioiodide is taken up from the blood and accumulates within thyroid follicular cells. About 20% of the iodide perfusing the thyroid is removed at each passage through the gland (23). The normal thyroid maintains a concentration of free iodide 20 to 50 times higher than that of plasma, depending on the amount of available iodine and the activity of the gland (24). This concentration gradient may be more than 100:1 in the hyperactive thyroid of patients with Graves' disease. The thyroid can also concentrate other ions, including bromide, astatide, pertechnetate, rhenate, and chlorate, but not fluoride (25;26).

Fig. 2-3: Radioautographs of rat thyroid sections. Animals received iodide shortly before sacrifice, and radioautographs of thyroid sections were coated with emulsion after being stained by the usual methods. The radioautographs indicated the presence of iodide primarily over the cells at these early time intervals. (From Pitt-Rivers, R.J., S.F. Niven, and M.R. Young, in Biochemistry, 90:205, 1964, with permission of the author and publisher.)

The protein responsible for iodide transport, the so-called sodium/iodide symporter or NIS, is located at the basolateral plasma membrane of thyrocytes (Fig. 2-4.). NIS-mediated I^- accumulation is a Na⁺-dependent active transport process that couples the energy released by the inward translocation of Na⁺ down to its electrochemical gradient to the simultaneous inward translocation of I^- against its electrochemical gradient. The maintenance of the Na⁺ gradient acting as the driving force is insured by Na⁺-K⁺-ATPase. NIS belongs to the sodium/glucose cotransport family as the SLC5A5 member. Iodide transport is energy-dependent and requires O₂. Ouabain, digitoxin, and other cardiac glycosides block transport in vitro (27;28). Iodide uptake by thyroid cells is dependent on membrane ATPase. During gland hyperplasia, iodide transport usually varies concordantly with plasma membrane Na⁺-K⁺-activated, ouabain-sensitive ATPase activity (29).

NIS cDNA was first cloned in rat FRTL-5 cells by Dai et al. (30). The rat NIS gene gives rise to a 3kb transcript with an open reading frame of 1,854 nucleotides encoding a polypeptide chain of 618 amino acids. The mature protein is a glycoprotein with an apparent molecular mass of 85kDa (31;32).It has 13 membrane spanning domains, with the carboxy terminus in the cytoplasm and the amino terminus located outside the cells (33). In the model of Levy et al. (34), a Na⁺ion first binds to the transporter which, in the presence of iodide, forms a complex that then transfers iodide and two Na⁺ ions to the cell interior.

The human NIS gene located on chromosome 19 (35) codes for a protein of 643 amino acids that is 84% homologous with rat NIS (36). The mouse NIS polypeptide chain (37) has the same size (618 amino acids) as the rat NIS. At variance with other species, three different transcripts are generated from the porcine NIS gene by alternative splicing (38); the main form encodes a polypeptide of 643 amino acids as human NIS.

Functional studies clearly show that NIS is responsible for most of the events previously described for iodide concentration by the thyroid. TSH stimulates NIS expression (39;40) and iodide transport (31;32). TSH exerts its regulatory action at the level of transcription through a thyroid-specific far-upstream enhancer denominated NUE (NIS Upstream Enhancer) that contains binding sites for the transcription factor Pax8 and a cAMP response element-like sequence. This original demonstration made on the rat NIS gene (41) has now been extended to human (42) and mouse (43) NIS genes. It has been suggested that TSH could also regulate NIS expression at post-transcriptional level (44). Data from TSH receptor-null mice (44-46) clearly show that TSH is required for expression of NIS. Moderate doses of iodide in the TSH-stimulated dog thyroid inhibit expression of the mRNAs for NIS and TPO, while not affecting that for Tg and TSH receptor (40) . The decrease in thyroid iodide transport resulting from excess iodide administration (escape from the Wolff-Chaikoff effect, see further) is related to a decrease in NIS expression (40;47). Both NIS mRNA and NIS protein are suppressed by TGFb, which also inhibits iodide uptake (48;49). Reviews focus on NIS and its functional importance (50;51).

Several mutations in the NIS gene causing defective iodide transport have been reported in humans (52-59). The most commonly found mutation corresponds to a single base alteration T354P in the ninth putative transmembrane domain of NIS (55). Site directed mutagenesis of rat NIS cDNA to substitute for threonine at residue 354 and transfection into COS cells lead to loss of iodide transport activity (60). Other mutations lead to truncated NIS (58) or to alterations of membrane targeting of the NIS protein (59) . NIS expression is increased in Grave’s disease and hyperactive nodules (61-63) and decreased in adenomas and carcinomas (64;65) appearing as cold nodules at scintigraphy.In hypofuctioning benign or malignant tumors, the impairment of iodide transport would result from both transcriptional and post-transcriptional alterations of NIS expression (66).Other tissues that concentrate iodide also show NIS expression, including salivary glands (67) and mammary glands (68;69).

Iodide supply of follicular lumen involves a two-step transport process: the active transport across the basolateral plasma membrane of thyrocytes by NIS and a passive transport across the apical plasma membrane. The protein(s) insuring the second step is (are) not yet identified. A potential iodide transporter has been proposed: pendrin (70;71). Pendrin, encoded by the PDS gene (72) and composed of 780 amino acids, is expressed in different organs including kidney, inner ear and thyroid. In the thyroid, pendrin is a 110kDA membrane glycoprotein (73), selectively located at the apical plasma membrane (74). Its activity as transporter of anions including iodide has been demonstrated in different experimental systems (71;75-77). Pendrin belongs to the SLC family under the reference SLC26A4. However, the implication of pendrin in thyroid iodide transport remains uncertain for several reasons. First, there is still no direct demonstration of a pendrin-mediated efflux of iodide from thyrocytes to the follicular lumen. Second, the genetic alterations of the PDS gene found in patients with the Pendred syndrome, which lead to a loss of the anion transport activity of pendrin and to a constant and severe hearing loss, only have a moderate impact on the thyroid functioning, generally a euthyroid goiter (78). Third, PDS knock-out mice (79) do not show any thyroid dysfunction. In summary, contrary to NIS for which the anion selectivity (25) corresponds to what was expected, the ion selectivity of thyroid pendrin remains to be elucidated. In the thyroid as in the kidney, pendrin could act primarily as a chloride/bicarbonate anion exchanger. Rather than pendrin, anoctamin-1/TEM 16A, a calcium-activated chloride channel, seems to be responsible for most of the iodide efflux accross the apical membrane of the thyrocytes (80).

Fig. 2-4: NIS-mediated transport of iodide. A, immunolocalization of the human NIS protein at the basolateral plasma membrane of thyrocytes in their typical follicle organization. B, schematic representation of the membrane topology of the NIS polypeptide chain deduced from secondary structure prediction analyses (33). C, transport of iodide from the extracellular fluid (or plasma) to the thyroid follicle lumen. The uptake of iodide at the basolateral plasma membrane of thyrocytes must be active; it operates against an electrical gradient (0 - 50 mV) and a concentration gradient, [ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle lumen should be a passive process, the electrical and concentration gradients being favorable.

Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and bound to tyrosyl residues in Tg. A significant proportion of intrathyroidal iodide is free for about 10-20 minutes after administration of a radioactive tracer (81), but in the steady state, iodide contributes less than 1% of the thyroid total iodine. A major fraction of the intrathyroidal free iodide pool comes from deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation. Some data suggest that iodide entering the gland by active transport segregates from that generated by deiodination of Tg within the gland (82;83). Once in the thyroid, iodide is organically bound at a rate of 50 to 100% of the pool each minute (24;84). The proportion of an iodide load that is bound varies little, despite wide shifts in daily intake. In contrast, NIS activity is sensitive to both iodine availability and TSH stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide available for hormonogenesis.

IODINE IN OTHER TISSUES

The thyroid is not the only organ to concentrate iodine; the others endowed with this capacity are salivary glands, gastric mucosa, mammary glands, and choroid plexus. Ductal cells of the salivary glands express NIS (67) . The plasma membrane of the mammary gland epithelium contains a NIS protein with a molecular mass different from that of thyroid NIS (~75 kDa vs ~90 kDa). In the mammary gland, NIS is processed differently after translation and subjected to regulation by lactogenic stimuli (68). It has been reported that over 80% of human breast cancer samples express this symporter. As it is absent in normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible target for radioiodine therapy (69). The thyroid, salivary glands, and gastric mucosa share a common embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is inhibited by thiocyanate, perchlorate, and cardiac glycosides. TSH stimulates transport only in the thyroid. An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk. Iodide concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit, but needs to be remembered for possible insights into pathways as yet undiscovered.

Iodine, particularly in the form of I₂, may enter additional metabolic pathways outside the thyroid. Rats administered I₂ orally showed much less circulating free iodide and much more iodine bound to proteins and lipids than did animals given iodide (85). In another comparison of I₂ versus iodide, administration of iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I₂. Additionally, I₂ decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased the former and had no effect on the latter (86).

THYROPEROXIDASE (TPO)

After concentrating iodide, the thyroid rapidly oxidizes it and binds it to tyrosyl residues in Tg, followed by coupling of iodotyrosines to form T₄and T₃. The process requires the presence of iodide, a peroxidase (TPO), a supply of H₂O₂, and an iodine acceptor protein (Tg).

Thyroperoxidase oxidizes iodide in the presence of H₂O₂. In crude thyroid homogenates, enzyme activity is associated to cell membranes. It can be solubilized using detergents such as deoxycholate or digitonin. The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely related porphyrin (87;88). Chemical removal of the prosthetic group inactivates the enzyme, and recombination with the heme protein restores activity (89). The apoprotein from human thyroid is not always fully saturated with its prosthetic group (90). Some congenitally goitrous children have poor peroxidase function because the apoprotein has weak binding for the heme group (90).

Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients with autoimmune thyroid disease (AITD), led to identification of TPO. These antibodies were found to react with proteins of 101-107 kDa and to immunoprecipitate thyroid peroxidase (TPO), thus identifying microsomal antigen as TPO (91-95). A monoclonal antibody to purified microsomal antigen or antibodies directed againt thyroperoxidase were then used to clone human TPO (96-98). Different laboratories then cloned TPO from various species: pig (99), rat (100), and mouse (101). Kimura et al. (96) cloned two different cDNAs of humanTPO.TPO1 coded for a protein of 933 residues and TPO2 was identical to TPO1 except that it lacked exon 10 and was composed of 876 residues. Both forms occur in normal and abnormal human thyroid tissue. The C-terminal portion of the proteins exhibits a hydrophobic segment (residues 847-871), likely corresponding to a transmembrane domain; thus, TPO has a short intracellular domain and most of the polypeptide chain is extracellular (Fig. 2-5A). TPO1 is active, but TPO2 appears enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface in transfected cell lines (102). Different degradative pathways exist for the two forms (103). Several other TPO variants resulting from exon skipping have been identified; they appear either active or inactive (104). Pig TPO contains 926 amino acids (99) ; mannose-rich oligosaccharide units occupy four of its five glycosylation sites (105).

Human TPO, which has 46% nucleotide and 44% amino acid sequence homology with human myeloperoxidase, clearly belongs to the same protein family. The TPO gene resides on chromosome 2p13, spans over 150 kbp, and has 17 exons (106). As NIS, Tg, and the TSH receptor (TSHr), TPO expression is controlled by the TSH cAMP pathway (107) through thyroid-specific transcription factors. These include TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 (108;109). Tg and TPO genes have the same binding sites for TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 in their promoters, and the genes for both have TTF-1/NKx2.1 sites in enhancer regions.

Inactivating mutations in the TPO gene are responsible for a subtype of congenital hypothyroidism characterized by thyroid dyshormonogenesis due to iodide organification defect. More than 60 annotated mutations have been reported; most of them result in total iodide organification defect with severe and permanent hypothyroidism (110;111).

TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes core glycosylation. TPO is then transported to the Golgi where it is subjected to terminal glycosylation and packaged into transport vesicles along with Tg (112) (Fig. 2-6). These vesicles fuse with the apical plasma membrane in a process stimulated by TSH. TPO delivered at the apical pole of thyrocytes exposes its catalytic site with the attached heme in the thyroid follicular lumen (113). TPO activity is restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the perinuclear part of the endoplasmic reticulum (114;115). Most of this intracellular protein is incompletely or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO has complex carbohydrate units. Glycosylation is essential for enzymatic activity (115). Chronic TSH stimulation increases the amount of TPO and its targetting at the apical membrane (116).

Fig. 2-5: Schematic representation of the membrane topology of Thyroperoxidase, TPO (A) and NADPH thyroid oxidase, ThOX (Duox) (B) at the apical plasma membrane of thyrocytes. C, hypothetical reaction scheme for TPO. H2O2 is presumed to oxidize the free enzyme with a loss of two electrons leading to the formation of complex I. Iodide binds to complex I, is oxidized and form complex II, which then reacts with a tyrosyl residue of Tg, Tyr-Tg.The newly-formed I0 and Tyr0-Tg free radicals interact to form MIT-Tg and the enzyme returns to its free state. I2 may be generated from two cxidized iodine atoms

H ₂ O ₂ GENERATING SYSTEM

By definition, a peroxidase requires H₂O₂ for its oxidative function. A large body of older work (reviewed in (117)) investigated possible sources using various in vitro models (117-120). It was already suggested in 1971 that H₂O₂ would be produced at the apical plasma membrane of the thyrocyte by an enzyme that requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway (121). Further biochemical studies showed that the enzymatic complex producing H₂O₂ for TPO is a membrane-bound NADPH-dependent flavoprotein (122-126). H₂O₂ produced by this NADPH-dependent protein is the limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is sufficient (127-129). In human thyroid, the H₂O₂ production and iodination process are stimulated by the calcium-phosphatidylinositol pathway (129). The quantity of H₂O₂ produced is important especially in stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes. While the activated leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr (130;131). Thus thyroid cells may be exposed to high doses of H₂O₂ and have to adapt to it by developing highly regulated generator and efficient protective systems.

More than twenty years passed between the initial biochemical studies and the cloning of Duox as the catalytic enzymatic core of the H₂O₂ thyroid generating system. By two independent molecular strategies Duox enzymes were uncovered from the thyroid. Starting from a purified fraction of pig thyroid membrane bound NADPH flavoprotein, the team of C. Dupuy isolated p138 ^Tox which turned out to be Duox2 lacking the first 338 residues (132). Simultaneously, De Deken et al cloned two cDNAs encoding NADPH oxidases using the strategy based on the functional similarities between H₂O₂ generation in the leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system would belong to the known gp91^phoxgene family and display sequence similarities with gp91^phox, now called NOX2. Screenings of two cDNA libraries at low stringency with a NOX2 probe enabled the isolation of two sequences coding for two NADPH oxidases of 1551 and 1548 amino acids respectively initially named Thox1 and Thox2 (133). The encoded polypeptides display 83% sequence similarity and are clearly related to gp91^phox (53 and 47% similarity over 569 amino acids of the C-terminal end) . The whole protein is composed of : a N-terminus ecto-sequence of 500 amino acids showing a similarity of 43% with thyroperoxidase (hence named Dual oxidase-Duox in the present terminology); a first transmembrane segment preceding a large cytosolic domain which contains two calcium binding EF-hand motifs; the C-terminal portion componed of six transmembrane segments, harbouring the four His and two Arg characteristic of the Nox family protein heme binding site and the conserved FAD- and NADPH-binding sites at the extreme C-terminal cytolic portion (Fig. 2-5B). Duox proteins are localized like TPO at the apical plasma membrane of the thyrocyte as fully glycosylated forms (~190kDa) and in the endoplasmic reticulum as high mannose glycosylated forms (~180kDa) (Fig. 2-6C).

Duox1 and Duox2 genes are co-localized on chromosome 15q15.3, span 75kb, have opposite transcriptional orientations and are separated by a ~16kb region (Fig. 2-6A). Duox1 gene is more telomeric, spans 36 kb and is composed of 35 exons; two first of them are non-coding. Duox2 spans 21.5 kb and is composed of 34 exons; the first being non-coding (134).

In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia, in oocytes (135-137) and Duox2 in gastrointestinal mucosa and salivary glands (138;139). Multiple functions are attributed to Duox enzymes: airway fuid acidification (140), mucin secretion (141), wound healing (142;143) and innate hoste defense (144-147) .

Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the gut (144;145;148;149) . Beside these killing mechanisms, Duox and H₂O₂ are certainly also involved in the interaction between host mucosa and bacteria to maintain mucosal homeostasis e.g. in bronchi and intestine (146;150). In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte would increase the efficiency of H₂O₂ producer-consumer system (151-153).

Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid differentiation marker. The proteins involved in the synthesis of thyroid hormones are expressed just after the thyroid precursor cells have completed their migration from the primitive pharynx and reached their final location around the trachea (154;155). This final morphological maturation begins in mouse with the expression of Tg at embryonic day 14 followed one day later (E15) by the expression of TPO, NIS, TSH receptor and Duox concomitant with the apparition of iodinared Tg (46;156).

Until 2006, the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell system for Duox correctly expressed at the plasma membrane in its active state. Several cell lines transfected with Duox1 and/or Duox2 showed Duox expression completely retained in the endoplasmic reticulum in their immature form without displaying any production of H₂O₂ (157). HEK293 cells transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent manner (158). The reconstitution of a Duox-based functional H₂O₂ generating system requires a maturation factor called DuoxA. The two human DuoxA paralogs were initially identified as thyroid specific expressed genes by in silico screenings of multiple parallel signature sequencing data bases (159). The two genes are located on chromosome 15 in the Duox1/Duox2 intergenic region in a tail to tail orientation, DuoxA1 facing Duox1 and DuoxA2 facing Duox2 (Fig. 2-6A.). DuoxA2 ORF spans 6 exons and encodes a 320 amino acid protein predicted to compose five transmembrane segments, a large external loop presenting N-glycosylation sites between the second and third transmembrane helices and a C-terminal cytoplamic region (Fig. 2-6B). DuoxA1 gene was initially annotated “homolog of Drosophila Numb-interacting protein: NIP” (160). Four alternatively spliced DuoxA1 variants have been identified (161). One of the most expressed transcript, DuoxA1α, is the closest homolog of DuoxA2 and encodes a 343 amino acid protein (58% identity of sequence with DuoxA2) adopting the same predicted structure.

In heterologous systems DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where they probably form complexes. Only the Duox1/DuoxA1 and Duox2/DuoxA2 pairs produce the highest levels of H₂O₂ as they undergo the glycosylation steps through the Golgi. Duox2/DuoxA1 pair does not produce H₂O₂ but rather superoxide anions and Duox1/DuoxA2 is unable to produce any ROS. In addition it has been shown that the type of Duox-dependent ROS poduction is dictated by defined sequences in DuoxA (162). This means that the Duox activators promote Duox maturation but also are parts of the H₂O₂ generating complex (163;164). Mice deficient in DuoxA maturation factors present a maturation defect of Duox, lacking the N-glycan processing, and a loss of H₂O₂ production. These mice develop severe goitrous congenital hypothyroidism with undetectable serum T4 and high serum TSH levels (165).

The reconstitution of this functional H₂O₂ producing system has been useful to measure and compare the intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma membrane under stimulation of the major signalling pathways active in the thyroid. It has been shown that the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium binding motifs. However, the two oxidase enzymatic activities are differently regulated after activation of the two main signalling cascades in the thyroid. Duox1 but not Duox2 activity is stimulated by the cAMP dependent cascade triggered by forskolin (EC50=0.1µM) via protein kinase A-mediated phosphorylation on serine 955 of Duox1. In contrast, phorbol esters, at low concentrations, induce Duox2 phosphorylation via protein kinase C activation associated with high H₂O₂ generation (EC50= 0.8nM) (166). These results suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H₂O₂ to TPO to oxidize iodide and couple iodotyrosines.

From in vitro and in vivo data it has been concluded that Duox-DuoxA constitutes the major if not the unique component of the hormonogenic thyroid H₂O₂ generating system. The bidirectional promoter allows the coexpression of Duox and DuoxA in the same tissue but the mechanisms regulating their transcription are not well and definitely characterized (167;168). It has been recently shown that Th2 cytokines, IL4 and IL13, up-regulate Duox2 and DuoxA2 genes in human thyrocytes through an activation of Jak-Stat pathway opening new perspectives for a better understanding of the eventual role of Duox in autoimmune diseases (169).

Defects in Duox and/or DuoxA were rapidly recognized possible causes of congenital hypothyroidism (CH) due to thyroid dyshormonogenesis in patients born with a hyperplastic thyroid or developing a goiter postnatally when T4 treatment is delayed after birth.

The first screening of mutations in Duox genes in 2002 was performed on 9 patients who had idiopathic congenital hypothyroidism with positive ClO4^- discharge (>10%), one with permanent and 8 with transient hypothyroidism (170). They were identified in the Netherlands by neonatal screening and followed up to determine the evolution of CH with the time. One of the patients with total organification defect (TIOD) presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a partial organification defect (PIOD). Of these last 8 patients 3 harboured heterozygous nonsense or frameshift mutations (Q686X, R701X, S965fsX994) meaning that a single defective Duox2 allele can cause haploinsufficency resulting in mild transient CH. It is noteworthy that this hypothyroid status was limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable in adulthood since adult heterozygotes in these families presented normal TSH serum levels. The only case with severe permanent CH was homozygous for a nonsense mutation (R434X= protein devoid of the catalytic core) leading to the conclusion at this time of a complete inability to synthesize thyroid hormone in absence of Duox2. No mutation was detected in Duox1.

With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to make the correlation between genotype and phenotype as initially described.

Indeed, subsequent studies have shown a link between biallelic Duox2 defects and PIOD. Patients with compound heterozygous missense (R376W) and a nonsense mutation (R842X), leading to a presumed non functional protein showed PIOD with mild and persistent hyperthyrotropinemia. This suggests that Duox1 can compensate at least partially for the defect in Duox2 (171). Varela et al.. described also two cases of permanent CH with compound heterozygous missense and nonsense or splicing mutations (Q36H and S965fsX994; G418fsX482 and g.IVS19-2A>C conducting to inactive proteins) responsible for congenital goiter with a PIOD (172).

The phenotype-genotype correlation suggested by the work of Moreno et al. is no longer clear. Maruo et al. described a series of transient CH characterized by biallelic defects in Duox2: in one family, four siblings were compound heterozygous for early frameshift mutations (L479SfsX2 and K628RfsX10) resulting in a presumed complete loss of Duox2 activity (not tested at this time). Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement therapy ceased to be necessary by 9yr of age (173). A French-Canadian patient with a transient CH initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense mutation (G1518S) inherited from the father and a deletion removing the part of the gene coding for the catalytic core of Duox2 inherited from the mother. In vitro test proved that the missense mutant protein was totally inactive (174). This case and others reported later provide further evidence that permanent or transient nature of CH is not directly related to the number of inactivated Duox2 alleles (175-177).

The first homozygous nonsense mutation in DuoxA2 (Y246X) that resulted in a non-functional protein tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a dyshormonogenic goiter (164;178). The mild phenotype can be explained by a partial maintenance of H₂O₂ production by Duox2/DuoxA1 as demonstrated in vitro. A high level of functional redundancy in Duox/DuoxA system could also explained the mild transient hypothyroidism in a patient with a novel biallelic DuoxA2 mutation and one allele of Duox2 and DuoxA1(179).

The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or by the activation of Duox1 or DuoxA1 in peculiar circumstances.

Fig 2-6: A, Localization of Duox and DuoxA genes on chromosomes 15q15.3. B, Schematic representation of the predicted structure of DuoxA (from (156). C, Immmunolocalization of human Duox and TPO at the apical membrane of the thyrocyte (upper: Duox immunostaining, middle:preimmune serum, lower:TPO immunostaining) (130).

THYROGLOBULIN (Tg)

Thyroglobulin is the most abundant protein in the thyroid gland; its concentration within the follicular lumen can reach 200-300 g/L. Its main function is to provide the polypeptide backbone for synthesis and storage of thyroid hormones (180). It also offers a convenient depot for iodine storage and retrieval when external iodine availability is scarce or uneven. Neosynthesised Tg polypeptide chains entering the lumen of the rough endoplasmic reticulum (RER) are subjected to core glycosylation, dimerise and are transferred to the Golgi where they undergo terminal glycosylation (Fig. 2-7). Iodination and hormone formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle colloid content.

Fig. 2-7: A polarized thyroid epithelial cell synthesizing soluble proteins, Tg (▲) and lysosomal enzymes (X) and membrane proteins, NIS (┴) and TPO (°). The polypeptide chain(s) generated by RER membrane-bound polysomes, enter the lumen of RER for the former and remain inserted into the RER membrane for the latter. Inside the lumen of RER, newly-synthesized proteins undergo core glycosylation and by interacting with chaperones acquire their conformation. Proteins are then transported to the Golgi apparatus (G), where terminal glycosylation and other post-translational reactions take place. In the Trans-Golgi network (TGN), mature proteins undergo sorting processes and are packed into transport vesicles. The vesicles carrying soluble proteins (inside the vesicle) and membrane proteins (as integral vesicle membrane protein) deliver them at the appropriate plasma membrane domain: the apical domain (1) and (2) or the basolateral domain (4). Vesicles carrying lysosomal enzymes (3) conveyed their content to prelysosomes or late endosomes (LE) and lysosomes (L). Apical plasma membrane proteins may reach their final destination by an alternative route involving a transient transfer to and then a retrieval and transport (*) from the basolateral membrane domain to the apical domain.

The Tg peptide chain derives from a gene of more than 200 kbp located on chromosome 8 in humans. The human Tg gene consisting of 48 exons (181) gives rise to a 8.5kb transcript that translates a 2,749 residue peptide (in addition to a 19-residue signal peptide) (182;183). The primary structure deduced from cDNA is also known for bovine, rat, and mouse (184-186). The biochemical traits of human Tg have been reviewed in (187). The N-terminal part of Tg has regions of highly conserved internal homology (10 motives of about 60 amino acids) which appears in several other proteins and are referred to as ‘thyroglobulin type-1 domains’. Such domains have been found to be potent inhibitors of cysteine proteases (188). This finding might be of importance, because these proteases are active in Tg proteolysis (see below). It has been suggested that this region of the Tg molecule may modulate its own degradation and hormone release (189). In the Tg-type 1 repeats, cysteine and proline residues are found in constant position; they may have an important role in the tridimensional structure of the protein. The proximal region of the C-terminal half portion of Tg contains five repeats of another type of cysteine-rich motives. The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide bonds, probably gives rise to peculiar structural constraints. The C-terminal portion of Tg is homologous with acetylcholinesterases (190). Because binding to cell membranes is one feature of acetylcholinesterases, perhaps Tg C-terminus has a similar role. It was reported that the acetylcholinesterase-homology region of Tg could function as a dimerization domain (178;191-193).Furthermore, three highly conserved thioredoxin boxes have been identified in mammalian Tg between residues 1,440 and 1,474; these boxes might be involved in disulfide bond formation leading to intermolecular cross-linking of Tg molecules inside the follicle lumen (194) . Tg gene expression is controlled by the same main thyroid-specific transcription factors that regulate synthesis of TPO (108):TTF-1/NKx2.1, TTF-2/FOXE1, and Pax-8 that bind at the same sites in Tg as they do in TPO. Hydrogen peroxide might be a regulatory factor of Tg expression, based on experimental work showing increased Tg promoter activity with reduced Pax-8 and TTF-1 (195-198). If substantiated, this proposal offers another point of integration between H₂O₂ generation and transcription of NIS, Tg and TPO genes, all of which being regulated by TSH.

Maturation of the Tg polypeptide chain begins while still on the RER. It undergoes core glycosylation and then monomers fold into stable dimers. Arvan and co-workers (199-204) have mapped this process and emphasize the role of molecular chaperones. The latter are essential for folding the new Tg molecules, and those that are folded improperly are not allowed to proceed further. The principal molecular chaperones are BiP, GRP 94, ERP 72, and calnexin. Only Tg molecules that pass this quality control system unscathed can proceed towards the secretory pathway. Glycosylation is a key event in Tg maturation. Carbohydrates comprise about 10% of Tg weight (205). Human Tg may contain four different types of carbohydrate units. The "polymannose" units consist only of mannose and N-acetylglucosamine. The "complex unit" has a core of three mannose residues with several chains of N-acetylglucosamine, galactose, and fucose or sialic acid extending from them. Both these types of unit are common in glycoproteins and are linked to peptide through an asparagine-N-acetylglucosamine bond. About three quarters of the potential N-glycosylation sites in human Tg are occupied, mostly with the complex unit (206). Two additional units have been found in human Tg; one contains galactosamine and is linked to the hydroxyl group of serine, the other is a chondroitin sulfate unit containing galactosamine and glucuronic acid (207) .

Failure in Tg folding can lead to disease as in the cog/cog mouse; these animals have a large thyroid with a distended ER and sparse Tg storage in follicles (208). Their Tg shows abnormal folding and decreased export from the ER in association with increased levels of several molecular chaperones. In the Tg cDNA of cog/cog mouse, Kim et al.(185) identified a single base substitution that changes leucine to proline at position 2,263. Correction of this defect by site-directed mutagenesis returned Tg export to normal in transfected cells. The cog/cog mouse is an example of endoplasmic reticulum storage disease (209). Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disorders (210). In each situation, the underlying defect appears to be a mutation in the coding sequence of exportable proteins. The ER retains the abnormal proteins, which cannot then proceed for further maturation. Several reports describe a similar pathogenesis for cases of congenital goiter and hypothyroidism in humans, although these are not as well characterized. Ohyama et al. (211) investigated a five-year-old euthyroid goitrous boy with high thyroidal radioiodine uptake, a positive perchlorate discharge test, apparently normal H₂O₂ generation and peroxidase activity in gland tissue, and low amounts of Tg in thyroid tissue overall, but large amounts in the RER. In another report, two hypothyroid goitrous sibs had a 138 bp segment missing between positions 5,590-5,727 in hTg mRNA, translating into a Tg polypeptide chain that lacked 46 residues (212). A third example described four subjects with congenital hypothyroid goiter from two unrelated families (213). Their thyroid tissue showed accumulation of Tg intracellularly with distension of the ER and large increases in activity of specific molecular chaperones, but with failure of Tg to reach the Golgi or the follicular lumen; this case was put forward as an ER storage disease similar to the cog/cog mouse (213).

Tg also contains sulphur and phosphorus. The former is present in the chondroitin sulfate and the complex carbohydrate units, although its form and role are not known (214). Several studies have reported phosphate in Tg, up to 12 mol. per mol Tg. Of this, about half is in the complex carbohydrate units, the remainder is present as phosphoserine and phosphotyrosine (215-217). This may relate to protein kinase A activity (218).

THYROGLOBULIN IODINATION AND HORMONE SYNTHESIS

The step preliminary to thyroid hormone formation is the attachment of iodine to tyrosyl residues in Tg to produce MIT and DIT. This process occurs at the apical plasma membrane-follicle lumen boundary and involves H₂O₂, iodide, TPO, and glycosylated Tg. All rendezvous at the apical membrane to achieve Tg iodination (Fig. 2-8).

Fig. 2-8: Iodination of Tg at the apical plasma membrane-follicle lumen boundary.The scheme does not account for the relative size of the intervening molecules

First, iodide must be oxidized to an iodinating form. An extensive literature has sought to identify the iodinating species, but the issue is still not resolved (see (219) for a detailed review). One scheme proposes that oxidation produces free radicals of iodine and tyrosine, while both are bound to TPO to form MIT which then separates from the enzyme (Fig.2-5C). Further reaction between free radicals of iodine and MIT gives DIT. Experimental studies by Taurog (219) and others suggest that the TPO reduction occurs directly in a two electron reaction. A second proposal, based on studies of rapid spectral absorption changes (88;220;221), is that TPO-I⁺ is the iodination intermediate and that the preferred route is oxidation of TPO by H₂O₂ followed by two electron oxidation of I^- to I⁺, which then reacts within a tyrosine. As a third possibility, Taurog (219) proposed a reaction between oxidized TPO and I^- to produce hypoiodite (OI^-), which also involves a two electron reaction. Whatever the precise nature of the iodinating species, it is clear that iodide is oxidized by H₂O₂ and TPO, and transferred to the tyrosyl groups of Tg. All tyrosine residues of Tg are not equally accessible to iodination. The molecule has about 132 tyrosyl residues among its two identical chains; at most, only about 1/3 of the tyrosyls are iodinated. As isolated from the thyroid, Tg rarely contains more than 1% iodine or about 52 iodine atoms.

The final step in hormone synthesis is the coupling of two neighbouring iodotyrosyl residues to form iodothyronine (Fig. 2-9). Two DIT form T4; one DIT and one MIT form T3. Coupling takes place while both acceptor and donor iodotyrosyl are in peptide linkage within the Tg molecule.The reaction is catalyzed by TPO, requires H₂O₂ (222-225) and is stringently dependent on Tg structure (226).The generation of the iodothyronine residue involves the formation of an ether bond between the iodophenol part of a donor tyrosyl and the hydroxyl group of the acceptor tyrosyl (Fig 2-10). After the cleavage reaction that gives the iodophenol, the alanine side chain of the donor tyrosyl remains in the Tg polypeptide chain as dehydroalanine (227-229). Observations both in vivo and in vitro show an appreciable delay in coupling after initial formation of iodotyrosines. A typical distribution for a Tg containing 0.5% iodine (a normal amount for iodine-sufficient individuals) is 5 residues MIT, 5 of DIT, 2.5 of T4 and 0.7 of T3 (180). More iodine increases the ratios of DIT/MIT and T4/T3, while iodine deficiency decreases them.

Fig. 2-9: Synthesis of hormone residues (coupling of iodotyrosines) in Tg at the apical plasma membrane-follicle lumen boundary. The scheme does not account for the relative size of the intervening molecules

Fig. 2-10: Possible coupling reaction sequence. Oxidation of iodotyrosines may produce iodotyrosyl radicals. The free radicals could combine to generate the iodothyronine residue (at the tyrosine acceptor site) and a dehydroalanine residue (at the tyrosine donor site), which in the presence of H2O converts into a serine

The distribution of hormone among several sites in the Tg molecule has been studied in a number of species (180;230-233). The most important is at tyrosyl 5, quite close to Tg N-terminus. It usually contains about 40% of Tg total T4. The second most important site is at tyrosyl 2554, which may contain for 20-25% of total T4. A third important site is at tyrosyl 2747, which appears favored for T3 synthesis in some species. Tyrosyl 1291 is prominent in T4 formation in guinea pigs and rabbits and very responsive to TSH stimulation. Incremental iodination of low iodine hTg in vitro, with lactoperoxidase as surrogate for TPO, led to the identification of the favored sites for iodination (234). Small increments of iodine go first to tyrosyl residues 2554, 130, 685, 847, 1448, and 5, in that order. Further addition increases the degree of iodination at these sites, iodinates some new tyrosyls, and results in thyroid hormone formation at residues 5, 2554, 2747, and 685, with a trace found at 1291, in that quantitative order. These data identified the most important hormonogenic sites in hTg, and also the favored sites for early iodination. The same work recognized three consensus sequences associated with iodination and hormone formation: i) Asp/Glu-Tyr at three of the four most important sites for hormone synthesis, ii) Ser/Thr-Tyr-Ser associated with hormone formation, including the C-terminal hormonogenic site that favors T3 in some species and iii)Glu-X-Tyr favoring early iodination, although usually not with hormone formation (Fig. 2-11).

Fig. 2-11: Diagram of the human Tg polypeptide chain; residue numbers refer to the human cDNA sequence; (a) sites forming T4 (sites A,B,D) (solid circles) and/or T3 (site C) (solid square); (b) early iodinated sites (solid triangles); (c) other iodinated sites (open triangles).

Identifying the donor tyrosyls has attracted considerable investigational interest over the past several decades. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of T4 makes them potential donor candidates (234). On the basis of in vitro iodination of an N-terminal cyanogen bromide Tg peptide, Marriq et al. (235) concluded that residue 130 was a donor tyrosine for the major hormonogenic site at Tyr5. This conclusion was challenged by Xiao et al. (236) in a similar in vitro system. A baculovirus system expressing the 1-198 fragment of Tg, either normal or mutated on tyrosyl residues, showed that iodination of a fragment containing tyrosyls only at residue 5, 107 and 130 formed T4 as did the intact normal peptide, but this fragment could also form T4 with substitutions at residue 5 or 130 (237). Dunn et al.(238) who incorporated 14C-Tyr into beef thyroid slices followed by in vitro iodination and trypsin digestion of the N-terminal portion of Tg localized pyruvate (as a derivative of dehydroalanine) to residue 130 by mass spectrometry. They proposed that Tyr130 was the donor tyrosine for the most important hormonogenic site at Tyr5. Gentile et al. (239) used mass spectrometry to identify a peptide containing dehydroalanine at tyrosine 1375 of bTg and proposed this tyrosine as the donor for the hormonogenic site at residue 1291. Donors for the other major hormonogenic sites have not yet been identified.

In addition to its role as component of the iodoamino acids, iodine is associated with cleavage of peptide bonds of Tg, at least in vitro (180). This has been attributed to generation of free radicals during oxidation (240). Exposure of Tg to reducing agents yields an N-terminal peptide of about 20-26kDa, depending on the animal species, that contains the major hormonogenic site of Tg (241). This peptide appears in parallel with iodination or may slightly precede it (242). Further addition of iodine cleaves the 26kDa further, to produce an 18kDa (on human Tg), an event that also occurs with TSH stimulation (242). Thus, iodination-associated cleavage appears to be part of the maturation of the Tg molecule. These discrete N-terminal peptides have been found in all vertebrate Tg examined so far (231).

The amount of iodine has important effects on thyroid hormone production (243). The initial reaction between TPO and H₂O₂ produces the so-called "compound I," which oxidizes iodide and iodinates Tg. Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid hormones. However, if excessive iodine is present, conversion to compound II does not take place, and hormone synthesis is impaired. (Fig. 2-12) Other iodinated compounds occasionally inhibit the thyroid. Thyroalbumin excited considerable interest several decades ago. This is an iodinated albumin, shown to be serum albumin that is iodinated in the thyroid (244). Occasionally, large amounts are found in certain thyroid diseases, including Hashimoto's thyroiditis (245), congenital metabolic defects (246), thyrotoxicosis (247) and thyroid carcinoma (248). In all these cases, there are abnormalities in thyroid structure which might explain the access of serum albumin to intrathyroidal iodination sites. However, in physiological conditions, serum albumin can reach thyroid follicle lumina by transcytosis i.e. basolateral endocytosis and vesicular transport to the apical plasma membrane (249). The thyroid also iodinates lipids and many different iodolipids have been described after high doses of iodide in vitro (250;251). Of particular interest is 2-iodohexadecanal (252;253). It occurs in the thyroid of several species following administration of KI, and its amount increases linearly with additional iodine, in contrast to iodination of Tg which eventually is inhibited by excess iodide. This compound inhibits the action of NADPH oxidase, which is responsible for H₂O₂ production (254;255). These findings suggested that iodination of lipids impairs H₂O₂ production and, therefore, decreases further Tg iodination. This is the most probable mechanism for the Wolff-Chaikoff effect (128).

Fig. 2-12: Demonstration of the Wolff-Chaikoff block induced by iodide in the rat. Animals were given increasing doses of stable iodide. There was at first an increase in total organification, but then, as the dose was increased further, a depression of organification of iodide and an increase in the free iodide present in the thyroid gland occurred.

HORMONE STORAGE

Tg molecules vectorially delivered to the follicule lumen by exocytosis accumulates to reach uncommun concentrations i.e. 0.3-0.5 mM.The mechanism operating such a “packaging” is unknown. Water and ion extraction from the follicle lumen might represent an active process leading toTg concentration. As the follicle lumen is a site of Ca⁺⁺ accumulation (256;257), the high degree of compaction of lumenal Tg might depend on electrostatic interactions between Ca⁺⁺ and anionic residues of Tg, which is an acidic protein. Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma membrane-lumen boundary (257-259), where TPO and H₂O₂ generating system reside. The mature Tg molecules, now containing MIT, DIT, T4 and T3, remains extracellular in the lumen of thyroid follicles. Turnover of intrafollicular material or so-called colloid varies greatly with gland activity. For normal humans, the organic iodine pool (largely in intrafollicular material), turns over at a rate of about 1% per day (14). When the turnover increases, less Tg is stored, and with extreme hyperplasia, none is evident and the entire organic iodine content may be renewed daily (14). In this situation, secretion of Tg and resorption of Tg (see below) probably occur at similar rates and only tiny amounts of intrafollicular material are present at any time.

Thyroglobulin as usually isolated from the thyroid is chiefly the 19S 660kDa dimer that has been glycosylated and iodinated. Iodination and hormone formation of Tg is more complex than generally thought because of the slow diffusion of molecules that are in a colloidal state in the follicle lumen. It has been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules (260;261). The diffusion coefficient of Tg which is about 26mm²/ sec in water would only be in the order of 10-100mm²/ hour in the thyroid follicle lumen. There is evidence for the presence of insoluble Tg in the form of globules of 20-120 microns, at a protein concentration of almost 600 mg/mL, in the lumen of thyroid follicles of different animal species (262). In human, about 34% of the gland Tg would be in this form (263). In pig, insoluble Tg contains more iodine than did the 660kDa Tg, and had virtually no thyroid hormone (264). Insoluble Tg has many internal crosslinks through disulfide bonds, dityrosine, and glutamyl-lysine bonds, the latter generated by transglutaminase (265). The formation of Tg multimers that probably results from oxidative processes might be limited by the presence of molecular chaperones such as the protein disulfide isomerase (PDI) and BiP in the follicle lumen (266).

THYROGLOBULIN ENDOCYTOSIS

To be useful, thyroid hormones must be released from Tg and delivered to the circulation for action at their distant target tissues. Depending on numerous factors including - the supply of iodide as substrate, the activity of enzymes catalyzing hormone formation, the concentration and physico-chemical state of Tg - the hormone content of lumenal Tg molecules varies to a rather large extent. Tg molecules newly arrived in the follicle lumen with no or a low hormone content would co-exist with “older” Tg exhibiting up to 6-8 hormone residues. The downstream processes responsible for the production of free thyroid hormones from these prohormonal molecules must therefore adequately manage the use of these lumenal heterogeneous Tg stores to provide appropriate amounts of hormones for peripheral utilization. One would expect to find i) control systems preventing excess hormone production that would result from the processing of excessive amounts of prohormonal Tg molecules and ii) checking systems avoiding the use of Tg molecules with no or a low hormone content.

Fig. 2- 13: Visualization of Tg endocytosis by in vitro reconstituted thyroid follicles obtained from porcine thyrocytes in primary culture. Purified porcine Tg molecules labeled by covalent coupling of fluorescein were microinjected into the lumen of a follicle. A and B, phase contrast and fluorescence images taken at the time of microinjection. C and D, fluorescence images of the top (C) and the bottom (D) of the follicle after 2hr of incubation. Fluorescently-labeled Tg is present inside thyrocytes.

The way the thyroid follicle proceeds to generate free hormones from stored hormone containing Tg molecules has been known for a long time. Tg molecules are first taken up by polarized thyrocytes (Fig. 2-13) and then conveyed to lysosomal compartments for proteolytic cleavage that release T4 and T3 from their peptide linkages. The first step represents the limiting point in the thyroid hormone secretory pathway. Over the last decade, there has been substantial improvement in the knowledge of the cellular and molecular mechanisms governing the internalization or endocytosis and intracellular transport of the prohormone, Tg. The evolution has first been to consider that it could proceed via a mechanism different from phagocytosis, also named macropinocytosis, evidenced in rats under acute TSH stimulation (reviewed in (267)). Results obtained in rats and dogs have been for a long time extrapolated to the different animal species including human. There is now a number of experimental data indicating that in the thyroid of different species under physiological circumstances, basal internalization of Tg, mainly if not exclusively, occurs via vesicle-mediated endocytosis or micropinocytosis (reviewed in (268)), while macropinocytosis results from acute stimulation (Fig. 2-14) (269;270).

Fig. 2-14: Schematic representation of the two modes of internalization of Tg; Micropinocytosis (on the right) and Macropinocytosis or phagocytosis (on the left). Intralumenal Tg stores potentially subjected to endocytosis are composed of (recently secreted) non-iodinated Tg, iodinated Tg (Tg-I) and iodinated Tg containing iodothyronine residues (Tg-Ith).Abbreviations are: CV, Coated Vesicle; EE, Early Endosome; LE, Late Endosome; L, Lysosome; Pp, Pseudopod; CD, Colloid Droplet; PL, Phagolysosome. The scheme on the right indicates the three possible routes of transport of internalized Tg molecules reaching the EE: transport to LE, recycling towards the follicle lumen and transcytosis i.e.transport towards the basolateral plasma membrane.

The internalization process starts with the organization of microdomains at the apical plasma membrane of thyrocytes; these microdomains or pits, resulting from the recruitment and assembly of proteins (clathrin, adaptins…) on the cytoplasmic side of the membrane, invaginate to finally generate coated vesicles after membrane fission. Lumenal Tg molecules, either free or associated to membrane proteins acting as Tg receptors, enter the pits and are then sequestrated into the newly-formed vesicles (267-269). Tg internalization via vesicle-mediated endocytosis is regulated by TSH (268). The vesicles lose their coat and, through a complex fusion process, deliver their content into a first type of endocytic compartments, the early apical endosomes (270) (Fig 2-15). In these compartments, Tg molecules probably undergo sorting on the basis of recognition of different physico-chemical parameters either linked or independent such as the hormone content, exposed carbohydrates, conformation of peptide domains… A step of sorting appears as a prerequisite for subsequent differential cellular handling of Tg molecules. It has been shown that internalized Tg molecules can follow different intracellular pathways. Part of Tg molecules are conveyed via a vesicle transport system to the second type of endocytic compartments, late endosomes or prelysosomes. This route ending to lysosomes corresponds to the Tg degradation pathway for the generation of free thyroid hormones. It is reasonable to think that Tg molecules following this route are the more mature molecules (with a high hormone content) but, this has not been firmly demonstrated. The other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport towards the apical plasma membrane (271) or via a two-step vesicular transport to the Golgi apparatus and then to the apical plasma membrane (272). Alternately, Tg molecules are transported and released at the basolateral membrane domain of thyrocytes via transcytotic vesicles (262;273); a process accounting for the presence of Tg in plasma. The orientation of Tg molecules towards one or the other of these three routes requires the presence of receptors. However, one route could simply convey Tg molecules that are not selected for entering the other pathways.

Receptors involved in Tg endocytosis may operate at the apical plasma membrane for Tg internalization and downstream in apical early endosomes for Tg sorting. The requirement and/or the involvement of apical cell surface receptors has long been debated. Most investigators now recognize that receptors are not needed for internalization since Tg is present at a high concentration at the site of vesicle formation. So, Tg molecules are most likely internalized by fluid-phase endocytosis and not by receptor-mediated endocytosis. On the contrary, if apical membrane Tg receptors exist, their function would be to prevent the internalization of sub-classes of Tg molecules (274;275). As it is not conceivable that internalized Tg molecules could enter the different intracellular routes, described above, at random, Tg receptors must exist in early apical endosomes. A detailed review on potential Tg receptors has been made by Marino and Mc Cluskey (276).

The first candidate receptor, initially described by Consiglio et al.(277;278) was later identified as the asialoglycoprotein receptor composed of three subunits (RLH1,2 and 3). This receptor binds Tg at acidic pH and recognizes both sugar moities and peptide determinants on Tg (279). As low-iodinated Tg molecules are known to have a low sialic acid content, this receptor could be involved in sorting immature Tg molecules for recycling to the follicle lumen. A second receptor, still not identified, named N-acetylglucosamine receptor (280;281), presumably located in sub-apical compartments, interacts with Tg at acidic pH; it could also act as a receptor for recycling immature Tg molecules back to the follicle lumen. A third receptor; megalin, has more recently been discovered in the thyroid and has been the subject of extensive studies yielding convincing data (276;282-285). Megalin is an ubiquitous membrane protein belonging to the LDL receptor family. It is located in the apical region of thyrocytes and its expression is regulated by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity.In vitro and in vivo data indicate that Megalin is involved in the transcellular transport or transcytosis of Tg molecules, possibly with a low hormone content (286).

From the properties and subcellular location of these receptors, one can propose an integrated view of the sorting processes that would operate in early apical endosomes. The asialoglycoprotein receptor and/or the less defined N-acetylglucosamine receptor would recognize immature Tg for recycling and megalin would interact with Tg subjected to apical to basolateral transcytosis. The remaining Tg molecules would enter, without sorting, the functionally important pathway i.e. the prelysosome-lysosome route.

Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg internalization. Pseudopods representing extensions of the apical plasma membrane project into the follicle lumen and pinch off to form a resorption vacuole known as colloid droplet (287) .The colloid droplets then deliver their content to lysosomes. Pseudopod formation is one of the earliest effects of TSH on the gland, evident within several minutes after administration (288;289). In most species but perhaps not in rat, TSH stimulates macropinocytosis through the activation of the cyclic AMP cascade (290;291).

Fig. 2-15: Transmission electron microscope observations of apical endocytic structures in thyrocytes. Top: coated pits at the apical plasma membrane. Bottom: an early endosome located in the apical region. Bars, 200 nm.

PROTEOLYTIC CLEAVAGE OF THYROGLOBULIN

Internalized Tg molecules that are conveyed to lysosome compartments are subjected to diverse hydrolytic reactions leading to the generation of free thyroid hormones and to complete degradation of the protein. Given its composition, Tg is likely the substrate for the different lysosomal enzymes: proteases, glycohydrolases, phosphatases, sulfatases.... Efforts have been made to identify proteases involved in the release of hormonal residues from their peptide linkage in Tg. Endopeptidases such as cathepsin D, H and L (292-299) are capable of cleaving Tg.

Initial cleavage would bring into play endopeptidases and resulting products would be further processed by exopeptidases. Dunn et al. (295) showed that cathepsin B has exopeptidase activity as well as an endopeptidase action (295;297). These investigators tested the activities of human enzyme preparations against the 20kDa N-terminal peptide from rabbit Tg, which contains the dominant T4 site at residue 5. Extended cathepsin B incubation produced the dipeptide T4-Gln, corresponding to residues 5 and 6 of Tg. The combination of cathepsin B with the exopeptidase dipeptidase I released T4 from this dipeptide, although lysosomal dipeptidase I alone was not effective. Thus, the combination of cathepsin B and lysosomal dipeptidase I was sufficient to release free thyroid hormone from its major site at residue 5. The exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg other than residue 5 (297). Thus, Tg probably undergoes selective cleavage reactions at its N- and C- terminal ends to release iodothyronines that are located nearby (297;300). Starting from highly purified preparations of thyroid lysosomes, Rousset et al. (301-303) have identified intralysosomal Tg molecules with very limited structural alterations but devoid of hormone residue. One may think that proteolysis of Tg occurs in two sequential steps; i) early and selective cleavages to release T3 and T4 residues and ii) delayed and complete proteolysis. The reduction of the very high number of disulfide bonds might be the limiting reaction between the two steps. The nature and the origin of the reducing compounds acting on Tg are not known. Noteworthy, the possibility of proteolytic cleavage of Tg inside the follicle lumen, before internalization, has been proposed (304-307) but not yet confirmed by other groups.

After Tg digestion, T4 and T3 must go from the lysosomal compartments to the cytoplasm and from the cytoplasm out of the cell to enter the circulation. It has been postulated for decades that thyroid hormones are released from thyrocytes by simple diffusion. There are many objections to this view (308). One of these comes from the chemical nature of iodothyronines; T4 and T3, which are generally considered as lipophylic compounds possess charges on both their proximal (amino acid side chain) and distal (phenolate) parts. As now known for the entry of thyroid hormones in peripheral target cells, the exit of thyroid hormones from thyrocytes probably involves membrane transporter(s). Details of hormone transport across the lysosomal membrane and then across the basolateral plasma membrane are unknown, including whether it is an active or passive process. At present, only a lysosomal membrane transporter for iodotyrosines has been reported (309;310). Nevertheless the role of newly cloned peripheral tissue thyroid transporters (311;312) in this process remains to be defined.

The type I and type II iodothyronine 5'-deiodinase is present in the thyroid (63;313;314) and deiodinate about 10% of T4 to T3. The extent of this intrathyroidal deiodination is increased when the thyroid is stimulated by TSH (315;316). Estimates of average normal secretion for euthyroid humans are 94-110 µg T4 and 10-22 µg T3 daily (317). The thyroid may also convert some T4 to 3,3'5'-T3 (reverse T3) within the thyroid. About 70% of the Tg iodine content is in the form of DIT and MIT, so this represents an important part of the intrathyroid iodine pool. Rather than lose it to the circulation, the thyroid deiodinates MIT and DIT and returns most of iodide to the intrathyroidal iodide pool. The responsible enzyme i.e. the iodotyrosine deiodinase is an NADPH-dependent flavoprotein with a estimated molecular weight of about 42kDa (318) and recently identified as DEHAL1(319-322). About 3-5 times more iodide is formed inside the gland each day by this deiodinase than enters the cell from the serum (14). The importance of the internal recycling of iodide is demontrated by congenitally goitrous subjects who harbour mutations in DEHAL gene (322) and cannot deiodinate iodotyrosines. These patients are successfully treated with large amounts of iodide (323). Some iodine is lost from the gland through inefficiency of its recycling by the iodotyrosine deiodinase (14;87;317;324). This leak may increase as the thyroid adapts to a high daily iodine intake (325), possibly as an autoregulatory process to prevent excessive Tg iodination. Much more iodide can be lost from diseased glands. Ohtaki et al. (87) found that some iodide leaks from all glands, including normal ones, but that the amount increases markedly with gland iodine content, presumably reflecting a dependence on dietary iodine intake. Fisher et al. (317) reported that about 38 µg iodide was released when the mean T4 secretion was 53 µg/day.

Among other products which are released or leak out from the thyroid, there is Tg (326;327). The secretion of Tg is clinically important. Its presence in serum can be detected by a routine assay and provides a sensitive (although not always specific) marker for increased thyroid activity. Attempts have been made to determine the biochemical characteristics of circulating Tg molecules in terms of iodine content (328), structural integrity (329) and hormone content (330). Serum levels are elevated in patients with hyperplastic thyroid or thyroid nodules including differentiated thyroid cancer. Tg measurement can identify congenital hyperplastic goiter, endemic goiter, and many benign multinodular goiters, but its greatest application is in the follow-up of differentiated thyroid cancer (331). Most papillary and follicular cancers retain some of the metabolic functions of the normal thyrocyte, including the ability to synthesize and secrete Tg. Subjects who have differentiated thyroid cancer treated by surgery and radioiodine should not have normal thyroid tissue left, and therefore, should not secrete Tg. If Tg is found in their serum, it reflects the continuing presence either of normal tissue, unlikely after its previous ablation, or of thyroid cancer. The depolarized cancer cells presumably secrete Tg directly in intercellular space. Tracking serum Tg levels is probably the most sensitive and practical means for the follow-up of such patients. It is more sensitive when the subject is stimulated by TSH. Until recently, this could only be done by withdrawal of thyroid hormone and consequent symptomatic hypothyroidism, but now recombinant human TSH can be administered to enhance the sensitivity of the serum Tg and thyroid scan (332-337).

CONTROL OF HORMONE SYNTHESIS

The most important controlling factors are iodine availability and TSH. Inadequate amounts of iodine lead to inadequate thyroid hormone production, increased TSH secretion and thyroid stimulation, and goiter in an attempt to compensate. Excess iodide acutely inhibits thyroid hormone synthesis, the Wolff-Chaikoff effect (243), apparently by inhibiting H₂O₂ generation, and therefore, blocking Tg iodination (127). A proposed mechanism is that the excess iodide leads to the formation of 2-iodohexadecanal (255), which is endowed with an inhibitory action on H₂O₂ generation.

TSH influences virtually every step in thyroid hormone synthesis and release. In humans the effects on secretion appear to be mediated through the cAMP cascade (see chapter 1) while the effects on synthesis are mediated by the Gq/phospholipase C cascade (338). Elsewhere in this chapter, we have mentioned instances of TSH regulation. To summarize, TSH stimulates the expression of NIS, TPO, Tg and the generation of H₂O₂ , increases formation of T3 relative to T4, alters the priority of iodination and hormonogenesis among tyrosyls and promotes the rapid internalization of Tg by thyrocytes. These several steps are interrelated and have the net effects of increasing the amount of iodine available to the cells and of making and releasing a larger amount and a more effective type of thyroid hormone (T3).

Anti-thyroid drugs are external compounds influencing thyroid hormone synthesis. The major inhibitory drugs are the thionamides: propylthiouracil and methimazole. In the thyroid, they appear to act by competing with tyrosyl residues of Tg for oxidized iodine, at least in the rat (219). Iodotyrosyl coupling is also inhibited by these drugs and appears more sensitive to their effects than does tyrosyl iodination.

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Primary Testicular Failure

Abstract

Primary testicular failure may result in endocrine failure, leading to testosterone deficiency or exocrine failure causing impaired spermatogenesis and subsequently male infertility. While some aspects of primary testicular failure are described in detail in separate chapters of Endotext.com, this chapter focuses on congenital or acquired anorchia, Leydig cell hypoplasia, and spermatogenic failure including germ cell aplasia (Sertoli cell only syndrome), spermatogenic arrest, hypospermatogenesis, and mixed atrophy. In addition, genetic causes for primary testicular failure are described such as numerical chromosome aberrations including Klinefelter syndrome, XX-Male syndrome, and XYY syndrome, structural chromosome aberrations of the autosomes or sex chromosomes, and Y chromosome microdeletions. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.

Key messages

Bilateral anorchia is defined as a complete absence of testicular tissue in genetically and phenotypically male patients.
Leydig cells hypoplasia is caused by inactivating mutations of the LH receptor.
Primary spermatogenic failure has to be considered as a description of certain histopathologic phenotypes, and not as a manifestation of single disease entities.
Several genetic causes for primary spermatogenic failure have been elucidated recently.
Modern management of patients with primary testicular failure caused by numerical chromosome aberrations such as Klinefelter syndrome can ameliorate symptoms of testosterone deficiency and – at least in some patients – can overcome infertility.
Up to date clinical guidelines are available for molecular diagnosis of Y chromosome microdeletions.
Novel technologies such as whole-genome sequencing will help to greatly increase the fraction of men suffering from primary testicular failure with a clear genetic diagnosis.

INTRODUCTION

The testis has an endocrine as well as an exocrine function. Endocrine testicular failure results in testosterone deficiency. In primary endocrine testicular failure, a decline in testosterone secretion (resulting in a condition termed hypoandrogenism) is caused by a deficiency or absence of Leydig cell function. Clinically relevant diseases described in this chapter are anorchia, Leydig cell hypoplasia and numerical chromosome abnormalities. Testicular dysgenesis is another cause for primary testicular failure that is described in depth in Endotext.com, Pediatric Endocrinology, Chapter 7: Sexual Differentiation. In contrast to primary endocrine testicular failure, secondary endocrine testicular failure is caused by absent or insufficient bioactivity of GnRH or LH (see Endotext.com, Endocrinology of Male Reproduction, Chapter 5: Hypogonadotropic hypogonadism and gonadotropin therapy).

The phenotype of primary exocrine testicular failure is male infertility. A comprehensive review on causes and treatment of male infertility is given in Endotext.com, Endocrinology of Male Reproduction, Chapter 7: Clinical management of male infertility. Cryptorchidism as a clinically relevant cause for primary exocrine testicular failure is discussed in Endotext.com, Endocrinology of Male Reproduction, Chapter 19: Cryptorchidism and hypospadias and testicular tumors as a cause and/or sequelae of testicular failure is discussed in Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects.

This chapter focuses on anorchia, germ cell aplasia, spermatogenetic arrest, hypospermatogenesis, numerical chromosome abnormalities, structural chromosomal abnormalities, as well as Y chromosome microdeletions causing primary exocrine testicular failure.

ANORCHIA

Bilateral anorchia is defined as complete absence of testicular tissue in genetically and phenotypically male patients. In unilateral anorchia testicular tissue is still present on the contralateral side.

Pure anorchia has to be differentiated from conditions with ambiguous and intersex genitalia (see Endotext.com, Pediatric Endocrinology, Chapter 7: Sexual Differentiation). A clinically important differential diagnosis is cryptorchidism and testicular atrophy where testicular tissue is still detectable (see Endotext.com, Endocrinology of Male Reproduction, Chapter 19: Cryptorchidism and hypospadias).

Congenital Anorchia

Bilateral congenital anorchia is rare; the incidence appears to be 1:20,000 males. Unilateral congenital anorchia is about 4 times as frequent.

As male differentiation of the genital tract and development of the penis and scrotum is dependent on the production of anti-Mullerian hormone (AMH) and androgens, the testis must have disappeared after initial activity in cases of bilateral anorchia. For the development of Wolffian duct structures, an ipsilateral testis must be present at least up to the 16th week of gestation ("the vanishing testis syndrome") (1). Intrauterine infarction of a maldescended testis or testicular torsion appears to be the major contributor to anorchia (2).

In patients with congenital bilateral anorchia serum gonadotropins are already elevated in childhood and rise to very high levels from the age of puberty onwards. Testosterone levels remain within the castrate range. In patients with suspected bilateral anorchia it is mandatory to rule out cryptorchidism, as cryptorchidism is associated with an increased risk for testicular cancer and should definitively not be overlooked (see Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects). Both the hCG stimulation test, that examines testosterone secretory capacity, and serum AMH measurement can be used for differential diagnosis. During hCG administration testosterone levels remain unchanged in patients with bilateral anorchia even after a 7-day period of stimulation, while a rise can be detected in patients with cryptorchidism (3). In comparison to the hCG test, measurement of AMH, which is undetectable in anorchia, has a higher sensitivity, but equal specificity for differentiation of bilateral anorchia from bilateral cryptorchidism (4; 5). Endocrine tests are not useful for differential diagnosis of unilateral anorchia. In these cases imaging techniques such as computer tomography or MRT and finally exploratory surgery or laparoscopy have to be applied.

Unilateral anorchia does not require therapy. In phenotypically male patients with bilateral congenital anorchia, testosterone substitution has to be implemented at the time of expected puberty. For psychological or cosmetic reasons, implantation of testicular protheses could be offered to the patient although these are often expensive. To date, there is no treatment of infertility in bilateral anorchia.

Acquired Anorchia

Surgical removal of both testes in patients with androgen-dependent prostate carcinoma is the most prevalent cause for bilateral acquired anorchia. Other reasons include unintended removal or devascularisation during herniotomy, orchidopexy or other testicular surgery, testicular infarction, severe trauma and self-mutilation. If only one testis is lost then fertility and testosterone production will normally be maintained by the remaining testis and no specific therapy is required. However, patients with a single testis require careful management when surgery is planned on the remaining testis.

The clinical appearance in patients with bilateral acquired anorchia depends on the time when testicular loss occurred. Acquired anorchia before puberty leads to the characteristic phenotype of male eunuchoidism and after puberty to the phenotype of post-pubertal testosterone deficiency (see Endotext.com, Endocrinology of Male Reproduction, Chapter 2: Androgen physiology, pharmacology and abuse).

Untreated acquired bilateral anorchia seems to have no effect on life expectancy, but clearly has an adverse effect on the quality of life (6). If both testes have been removed for therapeutic purposes, e.g. in a patient with prostate carcinoma, androgen supplementation is contraindicated. All other patients have to receive permanent testosterone substitution from the time of the expected onset of puberty in order to induce pubertal development, and in an adult immediately after testicular loss to maintain the various androgen-dependent functions.

LEYDIG CELL HYPOPLASIA

Leydig cell hypoplasia is a rare disease with an autosomal recessive pattern of inheritance and estimated incidence of 1:1,000,000. The Leydig cells are unable to develop because of inactivating mutations of the LH receptor that fails to provide the necessary stimulation of intracellular pathways. The underlying gene defect in Leydig cell hypoplasia was first described by Kremer et al (7) and various other defects have since been described (8–21). Men with Leydig cell hypoplasia present with very low serum testosterone and high LH levels. Leydig cell hypoplasia belongs to the group of the disorders of sex differentiation (DSD) and is currently classified as 46,XY DSD.

The phenotype is dependent on the extent of intrauterine testosterone secretion. Two types of Leydig cell hypoplasia have been described. Type I is the most severe form, resulting in a female phenotype of the external genitalia with blind ending vagina, primary amenorrhea, and absence of secondary sex differentiation at puberty. It is caused by inactivating mutations in the LH receptor that completely prevent LH and hCG signal transduction and thus testosterone production. Leydig cell hypoplasia type II is characterized by milder signs of androgen deficiency with a predominantly male habitus but signs of hypogonadism with micropenis and/or hypospadia. This milder form is derived from mutations of the LH receptor, which only partially inactivate signal transduction and retain some responsiveness to LH (16). Testicular histology reveals seminiferous tubules, whereas Leydig cells are not present or appear only as immature forms. Epididymides and deferent ducts are usually present, whereas the uterus, tubes or upper vagina are not found. In a patient with Leydig cell hypoplasia type II lacking exon 10 of the LH receptor, maternal hCG synthesized during pregnancy probably led to the development of a normal male phenotype, whereas LH was unable to stimulate the mutant receptor at the time of puberty (22; 23). HCG treatment of this patient was capable of inducing testosterone biosynthesis and complete spermatogenesis (22). This case, however, represents an exception. Therapy of 46,XY DSD with complete feminization requires both orchidectomy because cryptorchid gonads are prone to malignant degeneration, and estrogen substitution therapy.

Spermatogenic failure

Whereas endocrine testicular failure causes hypogonadism, spermatogenic failure - defined as exocrine testicular failure - leads to male infertility. Spermatogenic failure might be caused by hypothalamic, pituitary, or testicular disorders. A comprehensive review on causes and treatments of male infertility is given in Endotext.com, Endocrinology of Male Reproduction, Chapter 7: Clinical management of male infertility. Various testicular etiologies of spermatogenic failure may lead to the same histopathological pattern. In this sense, spermatogenic failure such as germ cell aplasia (Sertoli cell only syndrome), maturation arrest (MA) at different levels of early round spermatids, primary spermatocytes, or spermatogonia, and hypospermatogenesis have to be clearly differentiated from normal spermatogenesis. A key point is that primary spermatogenic failure has to be considered as a description of certain histopathologic phenotypes, and not as a manifestation of single disease entities.

GERM CELL APLASIA (SERTOLI CELL ONLY SYNDROME)

Germ cell aplasia or Sertoli cell only syndrome (SCO) is a histopathologic phenotype that was first described by Del Castillo et al. in 1947 (24). In complete germ cell aplasia the tubules are reduced in diameter, and contain only Sertoli cells but no other cells involved in spermatogenesis [Figure 1]. Germ cell aplasia can also be focal with a variable percentage of tubules containing germ cells, but in these tubules spermatogenesis is often limited in both quantitative and qualitative terms (25), and such cases should be referred to as hypospermatogenesis (see below). Germ cell aplasia or SCO is one common cause of non-obstructive azoospermia (NOA).

Figure 1. Germinal cell aplasia or Sertoli cell only Syndrome: Seminiferous tubules exhibit only Sertoli cells (SCO). Note the thickening of the lamina propria, focal hyperplasia of Leydig cells (hypLc) and interstitial infiltration of lymphocytes (ly). Primary magnification, x 20.

The lamina propria of SCO tubules is often found to be thickened due to increased collagen type IV and increased thickness of the basal lamina. The latter is associated with an overabundance of the beta2 chain of laminin and thought to be related to spermatogenic dysfunction (26).

In congenital germ cell aplasia, the primordial germ cells do not migrate from the yolk sac into the future gonads or do not survive in the epithelium of the seminiferous tubule. Anti-neoplastic therapy with radiation or chemotherapy may cause complete loss of germ cells. Other reasons include viral infections of the testes such as mumps orchitis. Germ cell aplasia can occur in maldescended testes.

Chromosomal abnormalities, especially microdeletions of the Y chromosome, are important genetic causes for complete germ cell aplasia (27). These deletions have been characterized and deletions in the AZFb or AZFb+c regions were identified to be important genetic causes of SCO and/or MA resulting in azoospermia (28). Sertoli cells, although showing normal histology, have an increased apoptotic index (29–31).

Several studies tried to identify other genetic risk factors which are associated with SCO. SEPTINS belong to a family of polymerizing GTP-binding proteins being required, for example, for membrane compartmentalization, vesicle trafficking, mitosis and cytoskeletal remodeling. SEPTIN12 participates in male infertility, especially SCO. Although no mutations were found in patients with SCO, 8 coding single-nucleotide polymorphisms (SNP1-SNP8) could be detected in these patients and the genotype and allele frequencies in SNP3, SNP4, and SNP6 were notably higher than in the control group (32). Most recently, Miyamoto et al. (33) analyzed the human LRWD1 gene whose translated protein mediates the origin recognition complex in chromatin which is critical for chromatin organization in post-G1 cells. Again, no mutations in SCO patients were found, but allele frequencies of two of three SNPs (SNP1 and SNP2) were notably higher compared to controls.

A hint to genetic risk factors leading to SCO was given by Tüttelmann et al. (34). They evaluated copy number variants (CNVs) in patients with severe oligozoospermia and Sertoli-cell-only syndrome and found that sex-chromosomal CNVs were significantly overrepresented in patients with SCO.

Diagnosis of germ cell aplasia can only be made by testicular biopsy. However, the testicular biopsy may not be representative in certain patients, as testicular sperm have been retrieved by testicular sperm extraction (TESE) in patients with apparently "complete germ cell aplasia" following a diligent review of the testicular histology (35). In addition, it has been demonstrated in a large consecutive series of bilateral biopsies from 534 infertile men that a marked discordance of spermatogenic phenotype pattern between both testes can be detected in about 28% of patients (36). Therefore, multiple testicular biopsies of both testes must be scrupulously screened before a diagnosis of complete germ cell aplasia can be made (37).

Patients with the complete form of germ cell aplasia are always azoospermic. Currently, there is no therapy for exocrine testicular failure of patients with complete germ cell aplasia. In general, testosterone production in the Leydig cells is not affected and patients are normally androgenized, and only few patients have hypoandrogenism requiring treatment.

Some patients have the appearance of complete germinal cell aplasia in some tubules but with complete spermatogenesis in adjacent tubules (sometimes called ‘focal’ germinal cell aplasia) while others have the appearance of an excess number of precursor germ cells in relation to the number of mature spermatids in the epithelium. Such cases have been described as incomplete or focal germinal cell aplasia which implies, perhaps falsely, a commonality between these disorders and those with complete germinal cell aplasia in all tubules.

SPERMATOGENIC ARREST

Spermatogenic arrest is also not a specific diagnosis for primary exocrine testicular failure, but a histopathological description of the interruption of normal germ cell maturation [Figure 2] at the level of a specific cell type including that of spermatogonial arrest [Figure 3], spermatocyte arrest [Figure 4], and spermatid arrest [Figure 5]). Sometimes, seminiferous cords/nodules with immature Sertoli cells can be found. These Sertoli cells still exhibit anti-muellerian hormone expression indicating their prepubertal state of differentiation. A definite diagnosis can only be made by multiple testicular biopsies.

Figure 2. Normal spermatogenesis: Seminiferous epithelium in stage I (I) and stage III (III) of spermatogenesis showing spermatogonia (sg), pachytene spermatocytes (p), round step 1 and 3 spermatids (rsd) and elongating step 7 spermatids (elsd). Primary magnification, x 40.

Figure 3. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of spermatogonia. Note multilayered spermatogonia (spg). Arrow: Sertoli cell nuclei. Primary magnification, x 40.

Figure 4. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of primary spermatocytes in pachytene stage (p). Primary magnification, x 40.

Figure 5. Arrest of spermatogenesis: Seminiferous tubule showing arrest of spermatogenesis at the level of early round spermatids (rsd). Note prominent multinucleated spermatid (mrsd). Primary magnification, x 40.

Meiotic arrest is regularly found in patients showing non obstructive azoospermia being considered as idiopathic, because no genetic or other origin can be detected. There are numerous studies showing lack of expression of several genes in meiotic maturation arrest compared to normal spermatogenesis. A major subgroup of patients lacks BOULE protein expression in primary spermatocytes, which is key factor of meiosis (38). The defect seems to be due to factor(s) upstream of BOULE being involved in the transcription and/or translation of BOULE. Heat shock protein levels are low or absent, such as heat shock transcription factor, Y chromosome (HSFY) (39) or HSPA2 that is involved in DNA mismatch repair (MMR) (40). SYPC3, a gene responsible for the synaptonemal complex is also involved in MMR and was found to be reduced. There is increasing evidence that alterations of the SYPC3 gene are involved in spermatocyte maturation arrest. Although expression of SYCP3 mRNA is found in patients showing normal spermatogenesis and spermatocyte maturation arrest, the lack of expression in men with spermatogonial arrest, Sertoli Cell Only syndrome, and testicular atrophy suggests negative effect on spermatogenesis and male fertility (41). However, data concerning the involvement of SYCP3 mutations related to spermatocyte arrest are inconsistent. A mutation analysis of the SYCP3 gene for 58 patients revealed only polymorphisms (42). Miyamoto et al. found a 1 bp deletion (643delA) resulting in a truncation of the C-terminal region of the SYCP3 protein in two of 19 azoospermic men with maturation arrest versus 75 patients showing normal spermatogenesis (43). Recently Stouffs et al. detected one change present in an evolutionary important functional domain of the SYCP3 gene in only one male patient that was absent in more than 200 controls (44).

MicroRNA-383 was shown to be down-regulated in maturation arrest (45). It was associated with a hyperactive proliferation of germ cells in patients with mixed patterns of maturation arrest, indicating that miR-383 functions as a negative regulator of proliferation. The authors concluded that abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor (46). There is a possible feedback loop between the fragile X mental retardation protein (FMRP) and miRNA-383, and FMRP acts as negative regulator for miRNA-383 functions, a loop that seems to be disturbed in maturation arrest (47).

Increased apoptotic index associated with spermatocyte maturation arrest was reported (29–31), data that correspond to the lack of expression of survivin, an inhibitor of apoptosis (48). These data correspond to the reduction of cyclin A, required for both the mitotic and meiotic divisions, in meiotic arrest (49).

In tubules showing meiotic arrest, there is also disturbance of the expression pattern of genes that are required for spermiogenesis. For example, BET (bromodomain and extra terminal) genes encode for transcriptional regulators and for histone-interacting chromatin remodelers. BRDT (bromodine testis specific), a key molecule participating in chromatin remodeling, is required for creation and/or maintenance of the chromocenter in round spermatids, a structure that forms just after completion of meiosis (for review see (50). The BRDT protein is localized in the nuclei of spermatocytes, spermatids, and ejaculated spermatozoa, and transcription is almost zero in primary spermatocytes of testes showing meiotic arrest (51). These data indicate that genes being important for postmeiotic spermiogenesis are already disturbed in the premeiotic stage.

In some patients with predominant round spermatid maturation arrest, the expression of cAMP Responsive Element Modulator (CREM) is significantly reduced or undetectable (52). Most recently, different expression of chromatin remodeling factors between normal spermatogenesis and round spermatid maturation arrest were found and suggest that impaired epigenetic information and aberrant transcription represents one reason for spermatid maturation arrest (53). Studies of the numerous mouse knock out models that display a spermatogenic phenotype, including sperm cell arrest, has contributed little of clinical relevance to the large number of men with idiopathic infertility. The possible role of several gene mutations and polymorphisms has been extensively investigated but no clear-cut genetic factor could be identified so far (54; 55). Spermiogenesis is a complex process with numerous different factors being involved. Thus it should be noticed that many factors are described and will be found to be related or responsible for spermatid maturation arrest, such as Krüppel-like factor 4 (KLF4), a transcription factor which is involved in many cellular and developmental processes including terminal differentiation of cells and carcinogenesis. A significant altered subcellular localization in arrested spermatids gives a first hint at a role for KLF4 during spermiogenesis (56).

Data concerning the topic of possible epigenetic alterations related to spermatogenic defects are rare. Khazamipour et al. analyzed the methylation status in the specific CpG island of the promoter region of MTHFR (Methylenetetrahydrofolate reductase) and found a significant hyper-methylation in 53% of the patients showing NOA compared to 0% of patients with obstructive azoospermia and normal spermatogenesis, indicating that hyper-methylation is specific and not due to a general methylation defect (57). Authors suggest that epigenetic silencing of MTHFR may be involved in azoospermic infertility. A similar study analyzing the CpG island containing tissue specific differentially methylated regions (TDMRs) in the VASA gene revealed significantly higher methylation in maturation arrest compared to normal spermatogenesis (58). Hyper-methylation associated silencing of PIWIL2 and TDRD1 was reported by Heyn et al. in human infertile patients showing maturation arrest (59).

Adiga et al. evaluated the expression pattern of a DNA methyltransferase (DNMT3B) which is important for germ cell methylation (60). Although they found a reduced number of DNMT3B positive primary spermatocytes in the case of bilateral maturation arrest, the few mature spermatids did not reveal any alterations of global methylation status.

Additionally, there may also be extratesticular factors such as long standing ischemia due to malformation of valves in spermatic veins responsible for maturation arrest (61). Secondary factors for spermatogenetic arrest are toxic substances (radiotherapy, chemotherapy, antibiotics), heat or general diseases (liver or kidney insufficiency, sickle cell anaemia) (62).

Testicular volume, FSH and inhibin B may be in their respective normal range, but may also be elevated or decreased. When these clinical parameters are normal, the differential diagnosis includes obstructive and non-obstructive azoospermia and this distinction made by diagnostic biopsy.

The arrest may be caused by genetic or by secondary influences. Genetic etiologies include trisomy, balanced-autosomal anomalies (translocations, inversions) or deletions in the Y chromosome (Yq11). It is likely that many genetic factors exist but have not yet been identified.

Complete arrest of spermatogenesis results in azoospermia. To date, there is no known therapy for uniform spermatogenic arrest (63).

HYPOSPERMATOGENESIS

The histological phenotype “hypospermatogenesis” shows complete spermatogenesis, but the number of elongating or elongated spermatids is moderately or severely reduced and the composition of the seminiferous epithelium is often incomplete because of missing generations of germ cells.

There are numerous reports showing functional impairment or alterations in seminiferous tubules showing hypospermatogenesis. Hypospermatogenesis is often associated with multinucleated spermatids indicating failure in spermiogenesis, or with so-called “megalospermatocytes” that are the morphological representation of missing synaptonemal complexes during meiotic prophase (64; 65). Whereas mitotic activity of spermatogonia is reduced (66), the apoptotic index indicating increased germ cell degeneration is elevated as shown by caspase immunohistochemistry (31) or TUNEL analysis (30). Both are also true in the case of maturation arrest at different levels of germ cell development.

Concentric spherical concrements deriving from the basal lamina are often found, when ultrasonographic examination of the testis reveals “microlithiasis”. These concrements may be associated with carcinoma in situ (syn: testicular intraepithelial neoplasia: TIN).

During spermiogenesis, protamine mRNA, being associated with the prognosis of successful ICSI therapy, is reduced in early round spermatids (67; 68). The histone to protamine transition during spermiogenesis is due the transcription factor CREM (cAMP responsive element modulator) and CREM activators. There are different isoforms functioning as activators and repressors and the expression pattern is related to impaired spermatogenesis (69–71).

Sertoli cell function is impaired, which has been described by Bruning et al. (72) by three dimensional reconstruction indicating functional dedifferentiation. This phenomenon, found to be associated with numerous aspects of Sertoli cell function, was later reviewed by Sharpe et al. (73). Most recently, Fietz et al. (74) could show a reduced mRNA expression of the androgen binding protein by quantitative RT-PCR. Huthaniemi et al. (75) found increased testosterone levels associated with androgen receptor CAG repeat length and because of a constant testosterone to estrogen ratio, authors suggested increased estrogen levels to be responsible for impaired spermatogenesis. Contrary data were reported by Nenonen et al. (76) who found a non-linear association between androgen receptor CAG repeat length and risk of male subfertility. This meta-analysis including almost 4000 patients revealed that androgen receptors with both either short or long repeats displayed lower activity than the receptors with repeats of median length. On a cellular level, Fietz et al. (74) analyzed androgen receptor mRNA of Sertoli cell populations associated with defined spermatogenic impairment using laser assisted cell picking and did not find any correlation of CAG repeat length to testicular histology or AR expression, suggesting factors other than CAG repeat to be responsible for severe spermatogenic impairment including mixed atrophy. This was also found by Hadjkacem-Loukil et al. (77) in a cohort of Tunesian azoospermic men showing Sertoli Cell Only syndrome or maturation arrest.

The lamina propria looks mostly unaffected in routine histological sections. However, functional defects resulting in a loss of contractility i.e. such as myosin heavy chain (MHY11) (78) or smooth muscle actin (79) were associated with hypospermatogenesis or mixed atrophy.

Functional dedifferentiation was found in Leydig cell hyperplasia and adenoma indicated by downregulation of the Leydig cell specific relaxin-like factor using in situ hybridysation and immunohistochemistry (80).

In most patients with hypospermatogenesis, testicular volume is reduced. FSH is elevated in most, but not all patients, with serum levels correlating positively with the proportion of tubules with germ cell aplasia (81). Several studies have demonstrated that inhibin B is a more sensitive and specific endocrine marker of hypospermatogenesis (82; 83). However, even the combined measurement of inhibin B and FSH provides no certainty concerning the presence or absence of sperm in multiple testicular biopsies (84; 85).

Mixed Atrophy

In most oligozoo- or azoospermic patients, testicular biopsy reveals a pattern of different spermatogenic defects in adjacent tubules: “mixed atrophy” being first described by Sigg (86): the simultaneous occurrence of seminiferous tubules includes SCO tubules or even only lamina propria (tubular shadows). This requires a detailed score-count analysis (35; 37). Additionally, functional mRNA or protein analysis of gene expression pattern described above can help to optimize the diagnosis of the underlying defects.

From a practical clinical perspective, the differentiation is important as patients with hypospermatogenesis or mixed atrophy may have azoospermia or varying degrees of oligoasthenoteratozoospermia, and sperm may be retrieved from testicular biopsies (TESE) (35). Pregnancies can be achieved with sperm retrieved by TESE that are injected into mature oocytes by intracytoplasmic sperm injection (ICSI). It has been suggested that residual sperm production could be improved by FSH therapy in incomplete germ cell aplasia. Clinical studies performed so far have demonstrated some increase in sperm concentration in the ejaculate and improvement of pregnancy rate (87; 88).

NUMERICAL CHROMOSOME ABERRATIONS

Klinefelter Syndrome

Harry Klinefelter first described this syndrome in 1942 as a clinical condition with small testes, azoospermia, gynecomastia and an elevated serum FSH (89). Only in 1959 was the chromosomal basis of the disorder elucidated as the chromosomal constitution with a supernumerary X-chromosome. Subsequently, the diagnosis of Klinefelter syndrome is made by chromosome analysis demonstrating the 47,XXY karyotype or one of its rarer variants.

The prevalence of Klinefelter syndrome is approximately 1 in 1,000 to 1 in 500 males (90). It is the most frequent form of primary testicular dysfunction affecting spermatogenesis as well as hormone production and is found in about 3% of unselected infertile men and >10% of men presenting with azoospermia (91; 92). It appears that at least half of the cases remain undiagnosed and untreated throughout life (90).

A non-mosaic 47,XXY karyotype is found in 80 - 90 percent of Klinefelter patients and mosaicism is seen in another 5 - 10 percent. The 47,XXY/46,XY mosaicism is most common. The 48,XXXY, 48,XXYY and 49,XXXXY karyotypes constitute 4 - 5 percent of all Klinefelter syndrome karyotypes, structurally abnormal extra X chromosomes are found in less than one percent of patients. Apart from karyotype analysis, molecular genetics methods can be used to quantify the number of X chromosomes, for example by quantitative PCR analysis of the androgen receptor gene located on the X chromosome (93).

The numerical aberration in non-mosaic 47,XXY is derived with equal likelihood from maternal or paternal meiotic error (94; 95). Most cases are caused by meiosis without X/Y or X/X recombination. Advanced maternal age seems to be a risk factor (90). It is not known whether the 47,XXY karyotype is slightly over-represented among spontaneous abortions and stillbirths. However, in contrast to many other aneuploidies, Klinefelter syndrome seems to be only a minor risk factor and most pregnancies result in a live-birth.

Patients with Klinefelter syndrome are usually inconspicuous until puberty. Interestingly the velocity of height gain can be increased in the pre-pubertal years. Men with Klinefelter syndrome tend to be tall (mean adult height is about the 80th percentile for the population) and to have relatively long legs compared to their overall height. Previously, the tall stature in KS was mainly thought to be a consequence of the hypogonadism, i.e. lower testosterone/estradiol levels not stopping long-bone growth by inducing epiphyseal growth plate fusion. However, more recent data comparing gonosomal aneuploidies support that increased body height is caused by excessive expression of growth-related genes. In this respect, the SHOX-gene is the leading candidate as it is located in the pseudoautosomal region and therefore present in three copies in Klinefelter men (96).

In most patients, early stages of puberty proceed normally. Post-pubertally the syndrome is characterized by small testes with firm consistency remaining in the range of 1 - 4 ml. Most patients with Klinefelter syndrome are infertile because of azoospermia. Testicular histopathology in adult men with Klinefelter syndrome displays various patterns. Classically, germ cell aplasia, total tubular atrophy or hyalinizing fibrosis and relative hyperplasia of Leydig cells are found. However, in some adult Klinefelter patients, foci of spermatogenesis up to the stage of mature testicular sperm can be detected ((97), and see below).

The degree of virilization varies widely. In early puberty, LH and FSH increase while serum levels of testosterone plateaus at or just below the lower limit of the normal range. After the age of 25, about 80% of patients have reduced serum testosterone levels and complain of decreasing libido and potency. On average, serum estradiol levels are high normal or may exceed the normal range. LH and especially FSH levels are exceedingly high, serum levels of inhibin B are very low or undetectable (98; 99).

During puberty, bilateral painless gynecomastia of varying degrees develops in about half of the patients. In a large Danish study covering 696 men with Klinefelter syndrome, no evidence for a substantial increase in the overall cancer rate was found (100). The risk of developing mammary carcinoma may be increased relative to normal men but remains a rare occurrence and routine surveillance is not recommended (100; 101). A significantly increased risk was found for the rare mediastinal malignant germ cell tumors, which occur preferentially at the age of 14 to 29 years (100).

The intelligence of Klinefelter patients is very variable. The group difference between boys with Klinefelter syndrome and controls amounts to 11 points in full scale IQ (92 versus 103), and deficits are observed primarily in verbal and cognitive abilities (102). Some of the young patients attract attention because of learning difficulties and school problems. They may fail to reach the level of achievement or professional expectations of their families (103; 104). Compared with their classmates, certain abnormal physical and psychological characteristics of the patients become obvious and they may become socially alienated. Higher-grade aneuploidy of the sex-chromosomes (48,XXXY, 48,XXYY and 49,XXXXY) is associated with mild to severe mental retardation while Klinefelter patients with chromosomal mosaicism (47,XXY/46,XY) may show very few clinical symptoms.

In general, the variability of the clinical features in patients with Klinefelter syndrome is related to the degree of androgenisation, which, in turn, partly depends on the pattern of inactivation of one copy of the androgen receptor gene. In particular, a significant genotype-phenotype association exists in Klinefelter patients and androgen effects on appearance and social characteristics are modulated by the androgen receptor CAGn polymorphism (105; 106).

Regarding infertility treatment, it should be noted that in rare cases sperm could be found in the ejaculate and, exceptionally, spontaneous paternity has been described (107). The rate of diploidy of sperm as well as disomy for gonosomes and autosomes has been reported to be increased in patients with Klinefelter syndrome, however, the majority of sperm appear to be normal (108–111). Almost two decades of experience with TESE/ICSI in patients with Klinefelter syndrome demonstrates that testicular sperm can be recovered in about 50% of the patients (112–115). Increasing age may be a negative predictive factor for successful TESE and some advocate to offer TESE and cryopreservation of tissue/spermatozoa already to teenaged patients. To what extent other factors such as previous testosterone treatment influence the chances of successful TESE remains under debate, as does the suggested treatment with drugs increasing FSH prior to TESE (116). So far, over 170 babies were born using testicular sperm for ISCI, all showing normal karyotype, although aneuploidies can be occasionally found by preimplantation or prenatal diagnosis (117). However, since the birth of normal children conceived by assisted reproductive techniques seems to be the rule (115), preimplantation diagnosis is not per se indicated. Based on indirect clues, it was postulated that 47,XXY spermatogonia are able to complete meiosis (118). However, Sciurano et al. nicely showed by fluorescence in situ hybridization (FISH) in testicular tissue of Klinefelter patients that all meiotic spermatocytes were euploid 46,XY(119). Fittingly, the common birth of children with normal karyotype suggests that the few sperm which can be found in patients with Klinefelter syndrome derive from the clonal expansion of spermatogonia with normal karyotype.

When testosterone serum levels are reduced, substitution with testosterone is necessary. To avoid symptoms of androgen deficiency, hormone replacement therapy should be initiated as early as needed. In particular, Nielsen et al. (120) showed that early testosterone replacement not only relieves biological symptoms such as anemia, osteoporosis, muscular weakness and impotence, but also leads to better social adjustment and integration. However, concurrent testosterone treatment severely reduces the chances of successful TESE and, therefore, the option of TESE should be considered before starting the first testosterone substitution and otherwise treatment should be stopped before the biopsy. Testosterone replacement must be considered a lifelong therapy in Klinefelter patients to assure quality of life. Usually gynecomastia is not influenced by hormone therapy. If it disturbs the patient, a plastic surgeon experienced in cosmetic breast surgery could perform a mastectomy.

XX-Male Syndrome

The XX-Male Syndrome is characterized by the combination of male external genitalia, testicular differentiation of the gonads and a 46,XX karyotype by conventional cytogenetic analysis. This disorder shows a prevalence of 1:9,000 to 1:20,000.

Applying fluorescence in situ hybridization or molecular methods it has been demonstrated that about 80% of XX-males have Y chromosomal material translocated onto the tip of one X chromosome (121). Translocation of a DNA-segment which contains the testis-determining gene (SRY = Sex Determining Region Y) from the Y to the X chromosome takes place during paternal meiosis (122). The presence of the gene is sufficient to cause the initially indifferent gonad to develop into a testis. The breakpoints and consecutively the size and content of the translocation seem to influence the severity of the phenotype (123).

Most SRY-positive patients are very similar to patients with Klinefelter syndrome. In general, however, 46,XX males are significantly shorter than Klinefelter patients or healthy men, resembling female controls in height and weight, which is in line with the recent view that the number of sex-chromosomes (most likely copies of the SHOX-gene) largely determines final height (96). The incidence of maldescended testes is significantly higher than that in Klinefelter patients and controls (124). The testes are small (1 - 4 ml) and firm, and endocrine changes of primary testicular failure with decreased serum testosterone and elevated estrogen and gonadotropin levels are observed. About every second patient develops gynecomastia. XX-males seem to have normal intelligence, however, exact data are lacking. Ejaculate analysis reveals azoospermia. The testicular histology of postpubertal SRY-positive XX males shows atrophy and hyalinization of the seminiferous tubules devoid of germ cells.

In SRY-negative XX-males (about 20% of XX-males), mutations in SOX9, RSPO1 or other candidate genes may be responsible for the sex reversal, but these are very rare and the mechanism underlying the majority of cases currently remains unclear (125). SRY-negative XX-males are generally less virilized than SRY-positive men and may show additional malformations of the genital organs such as maldescended testes, bifid scrotum or hypospadias (126).

Today, there is no therapy for infertility of men with XX-male syndrome. Patients with reduced testosterone production have to receive appropriate testosterone replacement therapy.

XYY-Syndrome

Most 47,XYY males have no health problems distinct from those of 46,XY males. The diagnosis relies entirely on the cytogenetic demonstration of two Y chromosomes with an otherwise normal karyotype. The non-mosaic chromosomal aneuploidy is caused by non-disjunction in paternal meiosis. Usually the finding is incidental, occurring when karyotyping has been undertaken for unrelated issues. The prevalence among unselected newborns appears to be 1:1,000.

Men with 47,XYY-syndrome have serum levels of testosterone and gonadotropins, as well as testicular volumes, comparable to those of normal healthy men. Most men with 47,XYY-syndrome have normal fertility. Onset of puberty seems to be delayed by 6 months, adult height is 7 cm in excess of the male population mean. The intelligence quotient lies within the normal range, but men score an average of ten points less than age-matched peers. Behavioral problems are more common in 47,XYY males, however, a history of violent behavior is exceptional (127; 128).

Most 47,XYY-men do not need any specific therapy. Men who achieve fatherhood can expect chromosomally normal offspring probably with the same likelihood as normal men. Nevertheless, to be safe, prenatal diagnosis can be offered.

STRUCTURAL CHROMOSOME ABERRATIONS

Structural chromosome abnormalities encompass alterations of chromosome structure that are detectable through light-microscopic examination of banded metaphase preparations as well as smaller, sub-microscopic deletions and duplications that are only detectable with molecular genetics (e.g. array Comparative Genomic Hybridization, aCGH). Structural rearrangements such as Robertsonian translocations, that also imply a change in chromosome number, are also regarded as structural abnormalities.

Structural anomalies of the autosomes are distinguished from anomalies of the sex chromosomes (gonosomes). Especially reciprocal and Robertsonian translocations, inversions, marker chromosomes, X and Y isochromosomes, and Y chromosomal deletions are of practical importance for andrology. When evaluating a structural chromosomal anomaly for clinical purposes, the distinction between balanced and unbalanced structural aberrations is pivotal. The former are characterized by a deviation from normal chromosome structure but without a net loss or gain of genetic material. If no important gene is disrupted at the breakpoints, balanced structural aberrations exert no negative effect on general health but may cause spermatogenic failure (oligo- or azoospermia) and independent of that, an increase in the risk for unbalanced karyotypes in the offspring (91; 129; 130).

In unbalanced structural chromosomal abnormalities, genetic material is either missing or there is an overall net excess of material in the cell. Unbalanced chromosomal aberrations may be incompatible with life and lead to abortion or cause a broad spectrum of disease. Exceptions are deletions of the Y chromosome that may limit reproductive functions selectively, and are therefore of importance in reproductive medicine (see below).

The majority of male individuals carrying structural aberrations is probably fertile and need no specific therapy. Conversely, men with impaired spermatogenesis show an increased prevalence of structural chromosomal abnormalities (129; 91; 92). Infertile patients with structural chromosomal aberrations may conceive naturally while more severe cases may require 'symptomatic' treatment modalities such as intracytoplasmic sperm injection, however, success rates may be lower than in couples with normal karyotypes (131). It should also be considered that unbalanced karyotypes of the embryo may result from balanced parental chromosomal anomalies (132). For any carrier of a structural chromosome abnormality who considers fatherhood by any means, genetic counselling is strongly recommended, and it should be obligatory prior to any infertility treatment (133; 134). It should be mentioned that in many countries karyotyping of men with idiopathic infertility and decreased sperm concentration is recommended prior to ICSI therapy although an evidence based screening threshold does not exist (135). The risk of spontaneous pregnancy loss, congenital malformations regularly associated with developmental delay as a result of an unbalanced karyotype in the offspring, options of prenatal and preimplantation genetic diagnosis, and - for certain aberrations - the possibility that other family members are also affected should be discussed with the patient.

Structural Aberrations of the Autosomes

Balanced autosomal anomalies may interfere with the meiotic pairing of the chromosomes and thus adversely affect spermatogenesis. These abnormalities often do not display a typical clinical phenotype. The presence and extent of disturbed fertility cannot be foreseen in individual cases. The same balanced autosomal aberration can have a severe effect on spermatogenesis in one patient and none at all in another patient. Even brothers with the same pathological karyotype can have widely differing sperm densities. So far no clinical or laboratory parameter in an infertile male is known which reliably indicates the presence of an autosomal structural anomaly. Therefore, in cases of unclear azoospermia or (severe) oligozoospermia, karyotyping is generally advised (135).

Translocations and other structural chromosomal aberrations can be either a de novo occurrence in the subject or inherited. Therefore, testing in family members should be encouraged, as the presence of a chromosomal aberration is regularly associated with a higher rate of abortion and the risk for the birth of a severely handicapped child.

Structural Aberrations of Sex Chromosomes

An intact Y chromosome is essential for the male reproductive system. The male-specific region of the Y chromosome (MSY) differentiates the sexes and comprises 95% of the chromosome length (136). The SRY gene is localized on the short arm of the Y chromosome and it influences differentiation of the embryonic gonad into the testicular pathway. The long arm of the Y chromosome contains areas responsible for establishing regular spermatogenesis.

When speaking of deletions of the Y chromosome, those of the short and the long arm must be distinguished (137). Short arm deletions of the Y chromosome that encompass the sex determining SRY gene result in sex reversal. Clinically, affected subjects appear as phenotypically female individuals with somatic signs of Turner's syndrome. If the deletion affects the long arm, the phenotype will be male. Loss of the heterochromatic part of the Y chromosome's long arm (Yq12) leaves general and reproductive health unaffected. Deletions of the euchromatic part of the Y chromosome's long arm (Yq11) may affect spermatogenesis, because Yq11 harbors loci essential for spermatogenesis (136).

In addition to deletions, a series of further structural anomalies of the Y chromosome are known. Pericentric inversions generally remain without consequence. An isodicentric Y chromosome is a more complex aberration nearly always occurring as a mosaic with a 45,X-cell line. The phenotype may be male, female or ambiguous. Patients with a male phenotype are usually infertile. These patients have an increased risk of developing testicular tumors (see Endotext.com, Endocrinology of Male Reproduction, Chapter 13: Testicular cancer pathogenesis, diagnosis and endocrine aspects). Reciprocal translocations between the Y chromosome and one of the autosomes are rare. In most cases, spermatogenesis is severely disturbed. However, several men with these aberrations have been reported as fertile. Translocations between the X- and Y-chromosomes occur in several variations; often the karyotype is unbalanced. The correlation between karyotype and clinical presentation is complex. The phenotype may be male or female; fertility may be normal or disturbed.

The X chromosome contains numerous genes essential for survival. Every major deletion of this chromosome has a lethal or severe effect in the male sex. Translocations between the X chromosome and an autosome usually result in disturbed spermatogenesis, whereas inversions of the X chromosome do not substantially affect male fertility.

Y CHROMOSOME MICRODELETIONS

The human Y chromosome is not only the dominant sex determinator, but plays an essential role in the genetic regulation of spermatogenesis (138). The long arm of the Y chromosome contains three partially overlapping but discrete regions that are essential for normal spermatogenesis (136; 139). The loss of one of these regions, designated as AZF (azoospermia factor)a, AZFb (P5/proximal P1), AZFc (b2/b4), and AZFbc (with two variants differing in the proximal breakpoint: P5/distal P1 and P4/distal P1) can lead to infertility (136). The deleted regions are usually of submicroscopic dimensions and are known as Y chromosomal microdeletions. Their prevalence in azoospermic men lies between 5 - 10% and between 2 - 5% in cases of severe oligozoospermia (140). Clearly, the frequency of Y microdeletions is related to the criteria by which men have been selected (141; 142), whereas ethnic differences might exist as well (143). Deletions of the AZFc region represent about 80% of all AZF deletions (143). The type and mechanism of deletions have been recently clarified and result from homologous recombination between retroviral or palindromic sequences (144). The AZFc region includes 12 genes and transcription units, each present in a variable number of copies making a total of 32 copies (145). The classical complete deletion of AZFc (b2/b4 deletion), removes 3.5 Mb, corresponding to 21 copies of genes and transcription units (146). Even more gene copies are removed by more extensive deletions (7.7 Mb and 42 copies removed in P5/distal P1 deletions; 7.0 Mb and 38 copies removed P4/distal P1 deletions) (147). It remains unclear if any of the genes of the respective regions are indeed pathologically relevant.

Clinically the patients present with severely disturbed spermatogenesis; endocrine testicular function may or may not be affected by the microdeletion as in other cases of spermatogenetic failure. Testicular histopathology varies from complete or focal Sertoli-cell-only syndrome (SCO) to spermatogenic arrest or hypospermatogenesis with qualitatively intact but quantitatively severely reduced spermatogenesis (148; 143). In azoospermic men, the presence of a complete deletion of AZFa seems to be associated with uniform germ cell aplasia (complete SCO), while a histological picture of SCO or spermatogenic arrest seems common in men carrying complete AZFb or AZFbc deletions. However, in exceptional cases, complete AZFb-deletions seem compatible with finding, albeit very few, spermatozoa (149; 150). Overall, the chances for successful sperm retrieval in carriers of complete AZFa as well as AZFb and AZFbc deletions has still to be considered virtually zero. On the other hand, men carrying complete AZFc deletions have severe oligozoospermia in about 50% of cases and in azoospermic carriers, successful TESE seems possible in about half of them (148; 151). No clinical parameter can help distinguishing patients with microdeletions of the Y chromosome from infertile men without microdeletion and, therefore, screening of all men with severe oligo- or azoospermia and without other causes is indicated (143; 135). It should be noted that Y chromosome microdeletions have also been described in proven fertile men (152).

A positive result of the analysis, which should be carried out according to the standard recommended by the current guidelines (153), provides a causal explanation for the patient's disturbed spermatogenesis. Beyond this, the test also has prognostic value, as TESE is possible in about 50% of men with AZFc deletion and every son of such a patient will carry the paternal Y chromosomal microdeletion and thereby inherit disturbed fertility (154). Hence, genetic counseling is indicated for all carriers of Y chromosomal microdeletions (133; 148).

Smaller deletions removing only part of the AZFc region have been identified as a polymorphism significantly associated with infertility, especially oligozoospermia (145). These so-called gr/gr deletions arise by the same mechanism (homologous recombination) and have been extensively studied in large groups of men in different countries. Overall, they are found in about 6.8% of infertile men but also in 3.9% of the controls and four meta-analyses have reported significant Odds Ratios, reporting on average 2-2.5 fold increased risks of reduced sperm output/infertility (55; 155–157). Although they represent a significant risk factor for male infertility, they should be regarded as a polymorphism and for the time being this type of diagnostics offers no advantage in male infertility workup. Concerns have been raised that a gr/gr (partial AZFc) deletion may expand to a complete AZFc deletion in the next generation and gr/gr deletions have also been reported as risk factor for testicular cancer (158; 159). Currently, however, no general agreement to advise routine testing has been reached (55; 157; 160; 153).

OUTLOOK: NEW TECHNOLOGIES, NOVEL GENETIC CAUSES

For many years, single candidate genes have been evaluated - usually by genotyping single nucleotide polymorphisms or sequencing - with the goal of identifying causal mutations for spermatogenic failure. Most of these approaches were, however, not successful most probably because 1) “male infertility” as well as “spermatogenic failure” are highly genetically heterogeneous and 2) selection of patient groups is often not stringent. Conversely, with the advances in genetic technologies, namely array-Comparative Genomic Hybridization (array-CGH) and whole-exome or even -genome sequencing, it is now possible to perform unbiased genome-wide analyses. These novel methodologies easily outperform the previous candidate gene approach which is illustrated by an increasing number of recent publications of so-called Copy Number Variations (CNVs), larger DNA regions that may be duplicated or deleted, as well as single genes causing spermatogenic failure. Examples are studies presenting CNVs on the autosomes as well as sex-chromosomes that are associated with azoo- or severe oligozoospermia (34; 161–163) as well as genes that are frequently mutated in specific phenotypes like meiotic arrest (163). In the near future, these novel technologies will help to greatly increase the fraction of men with a clear genetic diagnosis.

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Abnormalities of Female Pubertal Development

ABSTRACT

Puberty is the period of growth that bridges childhood to adulthood and results in physical and sexual maturity as well as the capacity for reproduction. Over half of pubertal timing is considered heritable. Significant pathology can result in both advanced and delayed puberty and can result in altered attainment of adult height, secondary sexual characteristics and reproductive capacity. The age for evaluation of precocious puberty has changed in the recent past due to greater understanding of the timing of pubertal development and important racial differences. The early detection of significant intracranial pathology underscores the importance of the workup in young girls with true precocious puberty, and the close follow up of girls in whom a brain MRI is not initially indicated. GNRH agonists have become a mainstay of therapy in girls with precocious puberty. The optimal method of delivery and age of cessation is not known, but increases in adult height and no obvious reproductive sequelae have been demonstrated. Unlike precocious puberty, the definition of delayed puberty has not changed in recent years, and large studies suggest that the most common diagnosis after evaluation is constitutional delay, however, this is more common in boys presenting with delayed puberty than girls. The most common diagnosis in girls with delayed puberty is gonadal failure. Advances in reproductive technologies have allowed women with Turner’s syndrome and MRKH to build their families. Among phenotypic women with all or part of a Y chromosome, gonadal extirpation is recommended, the timing of which varies with their genetic analysis which is the greatest predictor of risk for germ cell tumors. Molecular research and newer techniques of genetic analysis such as genome wide association studies and next generation sequencing have allowed the identification of genetic mutations that may be responsible for some of the complex diseases that cause both delayed and precocious puberty. For complete coverage of this and related areas in Endocrinology, visit the free online web-textbook, www.endotext.org.

Introduction

The pubertal process is the period of transitional growth bridging the childhood years and adulthood. The genetic blueprint housed within the genome of the individual has long before set in motion a number of critical processes. The end result is the maturation of a multitude of endocrine axes necessary for (1) secondary sexual development and, (2) the attainment of the immediate capacity for reproduction. Intrinsic to this reproductive maturation is yet another important process of puberty: (3) a secondary wave of skeletal growth and the attainment of adult stature. Abnormal puberty, whether premature or delayed, may adversely influence each of these events resulting in an untimely or altered ability for spontaneous secondary sexual development and spontaneous reproduction or abnormal growth.

In recent years numerous advances have been made in molecular medicine and the assisted reproductive technologies. The impact of these advances has had a tremendous effect on the care of patients with abnormal puberty by: changing the initial counseling provided to our patients; allowing for new treatments during the time of altered pubertal growth; and, providing reproductive options to individuals previously known to be infertile and some considered sterile. In addition, new insight about the physiology of puberty and the genetics of these disorders has accumulated. The focus of this chapter will be on our expanded knowledge of both the genotypes and phenotypes of the disorders presenting as abnormal puberty.

NORMAL PUBERTY FOR GIRLS (IT’S OCCURRING EARLIER!): A basis for the Definition of Abnormal Puberty

Onset of Normal Pubertal Landmarks

The first somatic change associated with the initiation of puberty in girls is an increase in growth velocity. It is during the initial increment in growth velocity that the first sexual sign of puberty occurs. The initial standards of puberty were published in approximately 1970 by Marshall and Tanner. These standards reported that in British girls thelarche (breast budding) developed at an average age of 11 years, followed by adrenarche, the appearance of pubic hair. After thelarche and adrenarche, growth velocity continues to increase and peak, a landmark termed the adolescent growth spurt. A peak height velocity of 9 cm/year is attained at that time. Subsequently, with near closure of the epiphyses there is a deceleration phase for growth. It is in this deceleration phase of growth that menarche occurs. It is often at least 5 years after menarche until most of menstrual cycles are ovulatory; clinicians cannot consider that puberty is normal until this reproductive mechanism is established as it represents the final step in maturation of the HPO axis.

The sequence and timing of pubertal development may vary by ethnicity. The classic description of the normal sequence of pubertal signs as published by Marshall and Tanner was taken from studies of British Caucasian girls not long after WW II.(1,2) They noted that breast development was the first sign of puberty occurring on average at 11 years of age in the British girls. In contrast, a study of African girls in the 1970s noted that for the majority of them adrenarche preceded thelarche.

Several larger studies conducted in the United States have given further insight regarding the timing of pubertal events and suggest that the age of puberty may be decreasing. (3,4) The Pediatric Research in Office Settings (PROS) data were taken from a cross-sectional study of 17,077 American girls of whom 9.5% were African-American and 90.4% were Caucasian. It should be noted that Hispanic girls were included in both African-American and Caucasian groups. Surprisingly, nearly 30% of the African-American girls had evidence of breast and/or pubic hair development at age 7 years and nearly 50% by age 8 years. For Caucasian girls, 15% had started puberty by age 8 years and nearly 40% by age 9 years. The mean ages for breast and pubic hair growth were 10.0 and 10.5 years for Caucasian girls, respectively, and 8.9 and 8.8 years for African-Americans, respectively. The average age of menarche for Caucasian girls remained unchanged at approximately 12.8 years with the African-American girls starting menstruation earlier and at a mean age of 12.16 years. (3) The PROS results may have been skewed slightly given the fact that inspection rather than palpation was utilized to determine thelarche. The NHANES studies did not collect onset of pubic hair or breast data in girls prior to the age of 12 years, centering their analyses on the timing of menarche and the attainment of completed puberty. (4)

In most studies taken from the US, an earlier time of menarche was reported when compared to the older data with ranges from 2 to nearly 5 months earlier depending on the ethnic group studied. While it is reasonable to consider that the original British normatives published by Marshall and Tanner are likely different from the heterogeneous American population at the end of the 20^th century, tremendous debate about the shortcomings and interpretations of these American data has continued over the last 10 years in a number of different forums. An expert panel overall agreed that the weight of the evidence supports a secular trend toward earlier breast development and menarche but not for other female pubertal markers. (5) Some evidence exists that malnutrition in certain socioeconomic groups of US children may currently be reversing this trend. (5)

Determinants of Normal Pubertal Growth

From conception to the fusion of epiphyses during the later stages of puberty, a number of maturational processes occur for formation and modeling of the skeleton. Intrinsic to somatic growth is the initial mesenchymal cell condensation and differentiation into cartilage that serves as a template for subsequent bone formation. Osteoblast differentiation occurs on the surface of this cartilaginous template and endochondral bone formation results when such differentiation occurs on calcified cartilage at the growth plate.

Genetic, environmental (i.e., nutrition), and hormonal determinants exist which are critical for the attainment of adult stature. The long held tenets that adult height is polygenic have been supported by genome-wide association studies for height (6). It has been estimated that 50 or more loci are associated with final adult stature (6-8). If all of these genes are functional, these parental-inherited growth genes determine the final adult height attained by an individual. Minimal changes by any number of these genes may result in height variation within the predicted height distribution. One can estimate this height by a calculation of mid-parental height. For females this is determined by subtracting 13 cm from the father’s height, adding this to the mother’s height in cm and then dividing by 2.

Under pathophysiologic situations, an individual may be taller or shorter than would be dictated by parental height determinants. Sometimes these differences are genetically determined and in other situations abnormal hormonal influences alter an otherwise intact genetic predisposition, and in other cases environmental factors play a role.

Genetic Influences of Growth

Some statural genes are present on both X and Y-chromosomes with Y individuals being taller than X individuals. From tallest to shortest one can generalize the following: XYY > (taller than) XY > (taller than) XXX > (taller than) XX > (taller than) X individuals. A few genes have been implicated in these differences. One set of genes, the SHOX genes, exist on the distal X chromosome. (9-12) Mutations have resulted in short stature and deletion of this locus is associated with short stature in Turner syndrome (45,X). (9)

Hormonal Determinants of Growth (Some gene mediated)

No doubt, a normal endocrine environment critically influences bone growth. For example it is essential that intact and normal growth hormone and thyroid hormone production, among others, be present. This is demonstrated by the fact that growth hormone and thyroid hormone deficiency separately result in short stature until corrected. (13) Growth hormone excess results in such conditions as a gigantism and acromegaly.

In addition to these known growth-promoting hormones, sex steroids are essential for mediating the pubertal growth spurt and attainment of final adult stature. Premature sex hormone production in children with congenital adrenal hyperplasia causes premature epiphyseal growth and fusion: thus, tall as children and short as adults. Early onset precocious puberty similarly causes premature pubertal growth with the risk of short adult stature unless corrected. The lack of pubertal development (delayed puberty) allows for continued long bone growth since the epiphyseal centers remain open longer than normal. Usually, in these situations, growth is normal until the expected age onset of puberty and the growth spurt is not noticed; however, linear growth continues in the absence of epiphyseal closure. This results in eunuchoid body proportions: an arm span which exceeds the height by more than 6 cm and disproportionately long legs.

While it had always been accepted that estrogen mediates pubertal bone growth in females, it was not until this era of molecular medicine that it was determined that estrogen and not testosterone mediates the same function for males. Inactivating mutations in either the estrogen receptor gene or the aromatase gene (preventing conversion from androgens to estrogens) in males have resulted in lack of normal bone growth at puberty and lack of epiphyseal closure with resultant tall stature (i.e., taller than predicted). (14-17) These findings establish that estrogen is essential for initiation of pubertal growth, closure of the growth plate, and augmentation accrual of bone during puberty. The presence of both alpha and beta estrogen receptors have been identified in the growth plate and studies are underway to understand the exact mechanism of estrogen action. (18)

DEFINITION OF ABNORMAL PUBERTY

The classic definitions of abnormal puberty, whether premature or delayed, are based on timing that is considered to be 2.5 standard deviations removed from the mean. Previously, the definition of precocious development for girls was the appearance of secondary sexual development before the age of 8 years, an age felt to represent 2.5 standard deviations earlier than the mean.

Revised recommendations have been made based on the findings of the PROS Network. (19) These guidelines propose that precocious puberty be defined by the presence of breast or pubic hair development before age 6 years in African-American girls and age 7 years in Caucasian girls.

However some experts disagree with the PROS recommendations. A few girls with puberty starting between 6 and 8 years of age for African Americans and between 7 and 8 for Caucasians were initially reported with endocrine or CNS pathologic etiologies of early puberty. As a result, concerns emerged that the PROS definitions may miss significant pathology and that strict enforcement of the new guidelines will lead to missed diagnoses. (20-22) Data suggest that between 2-9% of girls in this age group will have underlying pathology and 1% will have a tumor. (23,24) Missed diagnoses have included CNS tumors, neurofibromatosis, hypothyroidism, congenital adrenal hyperplasia, and hyperinsulinism. Thus, for such children beyond the recommended age of evaluation with presenting symptoms of precocious puberty, a complete history and physical exam are warranted to ensure that a serious underlying condition is not missed. Some experts recommend a bone age evaluation and careful longitudinal follow-up for girls younger than age 8 years that do not fall into the PROS guidelines for evaluation for precocious puberty. (22)

Recommendations based on the findings of the PROS Network have not been made for revising the definition of delayed puberty in girls as they have for precocious. As such, the absence of thelarche by age 13 years for girls signifies an abnormality, and remains the definition of pubertal delay. The classic definition for delayed menarche, i.e., primary amenorrhea, has been the absence of menarche by age 15 or 16 years, which is approximately 2.5 to 3 standard deviations from the mean, respectively.

While some patients present strictly with the absence of the onset of pubertal development, others have abnormalities in the tempo and sequence of puberty that has seemingly begun on time. Menarche usually occurs within 3 years of thelarche, when most girls have tanner stage 4 breast development. The American Academy of Pediatrics and the American College of Obstetricians and Gynecologists have jointly published guidelines that recommend evaluation of delayed puberty if menarche does not occur within 3 years of thelarche. (25)

These guidelines also recommend evaluation of girls with the following characteristics:

No breast development by age 13 years (delayed puberty)
Absence of menarche by age 14 years in the presence of hirsutism or history or exam suggestive of eating disorder or excessive exercise or an outflow abnormality
Absence of menarche by age 15 years.

Age definitions should be seen only as general guidelines. Rather than require a young woman meet the strict definitions of menarche by age 15 or 16 years to initiate an evaluation for delayed puberty, it has been suggested that all adolescents be followed annually throughout the pubertal process. (26)

For example, if a young woman presents concerned because of no menses at age 14 years, some of the major etiologies of primary amenorrhea could be recognized at an office visit without adding any significant costs. Screening at an age prior to 15 years should, as discussed in the previous paragraph, include screening for eating disorders and consideration of an excessive androgen disorder such as polycystic ovary disease. Exclusion of outflow tract disorders such as vaginal agenesis or imperforate hymen / transverse vaginal septum would require gentle pelvic examination. The physical exam should also be directed to identify findings that are typical of some associated endocrinopathies or syndromes such as gonadal dysgenesis. It would be better to begin a partial evaluation (i.e., FSH level and use of growth velocity curve) during earlier adolescent years at the time that abnormalities are first suspected than it would to wait until these young women are significantly different from their peers. No doubt, adolescence is one of the most difficult time periods in growth and development. It is potentially very harmful for an individual’s psychosexual development to allow significant delays in secondary sexual development or onset of menses to continue without evaluation, treatment and appropriate counseling. Young women are particularly likely to be worried about delayed breast development.

PRECOCIOUS PUBERTY

Overview

The overall incidence of sexual precocity among American children has been estimated to be between 1:5,000 to 1:10,000. (27) The female to male ratio is approximately 10:1. Early activation of pulsatile gonadotropin-releasing hormone (GnRH) secretion is the most common mechanism of precocious puberty; usually it is idiopathic but it can be from serious conditions such as hypothalamic tumors. While the classic definition of sexual precocity is the appearance of secondary sexual characteristics before the age of 8 years in girls, newer guidelines as discussed above suggest that puberty is not considered precocious unless it occurs prior to age 6 years for African-American girls or age 7 years for Caucasian girls. (19) However, many pediatric endocrinologists in the United States routinely evaluate all girls with precocious development prior to the cutoff at age 8 years (28). As discussed above, even when puberty occurs between ages 6-7 and 8 years, it is important to consider evaluation of all children. (20-22) The child may be suffering from a serious CNS disorder associated with precocious puberty. (21)

Long term implications of early puberty include an increased risk of breast cancer, metabolic diseases (e.g., type 2 diabetes, obesity), endometrial cancer and cardiovascular disease. (29,30) In addition, psychosexual maturation remains concordant with chronological age, and unfortunately early physical sexual maturation at any age places these young girls at a high risk for sexual abuse. Clinicians should routinely screen children with early development for sexual abuse. Direct questioning in age appropriate language should be used and the history should include questions about behavioral markers including new onset bedwetting, nightmares, or other behavioral issues. It is thus important not only to make a reasoned judgment as to when to initiate an evaluation, but also to institute the appropriate therapy and support to prevent these potential long-term sequelae, even in selected girls who fall outside the new recommendations. It is also prudent to remember that early maturing girls, who may not “fit” the criteria of having premature puberty, may elect to engage sooner in coitus and other risk taking behaviors such as drugs than later maturing girls.(31,32)

Precocious puberty represents the appearance of the secondary sexual characteristics from increased sex steroid production. This increase may be secondary to aberrant gonadotropin stimulation or intrinsic disease of the ovary or adrenals. Many terms have been used to describe the types of precocious puberty, and some are less used in contemporary literature.

True precocious puberty, also known as complete precocious puberty, refers to puberty that appears early and either progresses through each of the pubertal landmarks including menarche or, in the absence of treatment, would likely progress through each of these stages. In the majority of children presenting for precocious development this early evidence of puberty is not the result of true precocious puberty and will halt or even regress; treatment is unnecessary (33). Classically a GnRH challenge test that demonstrated a pubertal response of gonadotropins (i.e., LH response > FSH response) was the hallmark of this diagnosis. The usual ability to suppress pubertal development with GnRH agonists remains the hallmark of treatment.

Incomplete precocious puberty refers to the appearance of one phase of the pubertal process: thelarche, adrenarche, or menarche. Isolated precocious thelarche, isolated precocious adrenarche, and isolated menarche are the three forms of incomplete precocious puberty.

Sexual precocity has been further categorized according to whether the pubertal signs are concordant or discordant with the sex of the individual: isosexual precocity referring to early sexual development consistent with the sex of the individual (i.e., feminization of a female); heterosexual or contrasexual precocity indicating precocious pubertal development that is limited to those physical signs not characteristic for the sex of the individual when presenting as isolated findings (i.e., virilization of a female). GnRH dependent and GnRH independent precocious puberty (GIPP) refer to those causes of precocity that are or are not secondary to GnRH production. Central precocious puberty (CPP) refers to precocity of CNS origin.

A summary of the causes of sexual precocity is presented in Table I below, followed by a numeric breakdown of the frequency of occurrence of these disorders in Table II.

Table I. Classification of Female Precocious Puberty

I. Complete isosexual precocity (true precocious puberty: gonadotropin dependent)

A.    Idiopathic

B.    CNS lesions: Hamartomas, Craniopharyngioma, etc

C.    Primary hypothyroidism

D.    Post treatment for CAH

E.     Genetic

II. Incomplete isosexual precocity (GnRH independent)

A.    Isolated precocious thelarche

B.    Isolated precocious menarche

C.    Estrogen-secreting tumors of the ovary or adrenals in girls

D.    Ovarian cysts

E.     McCune-Albright syndrome

F.     Peutz-Jeghers syndrome

G.    Iatrogenic

III. Contrasexual precocity (Isolated virilization)

A.    Isolated precocious adrenarche

B.    Congenital adrenal hyperplasia

C.    Androgen-secreting ovarian or adrenal neoplasm

D.    Iatrogenic

Table II. Numeric breakdown of etiologies for precocious puberty in a large series of girls (N=438) evaluated from 1988-1999 by the classic definition (pubertal onset < 8 years) (24)

I. Central Precocious Puberty 428 (97.7%)Incompletely Evaluated 124Completely Evaluated 304 Idiopathic 226 (74.4%)

CNS Pathology 56 (18.4%)

Hydrocephalus 11 (19.6%)

Encephalocele 2 (3.6%)

Neurofibromatosis 3 (5.4%)

Encephalitis 1 (1.7%)

Intracranial hemorrhage 1 (1.7%)

Hypothalamic hamartoma 7 (12.5%)

Pituitary microadenoma 5 (8.95%)

Optic chiasma astrocytomas 3 (5.4%)

Optic chiasm glioma 1 (1.7%)

CNS Vascular Malformation 1 (1.7%)

Other miscellaneous CNS disorders/lesions 21 (37.5%)

(100%)

Coincidental/Associated Disorders 22 (7.2%)

(100%)

II. GnRH Independent (GIPP) 10 (2.3%)

McCune Albright syndrome 3 (30%)

Ovarian “hyperfunction”/

follicular cyst 4 (40%)

Ovarian tumors 3 (30%)

Juvenile granulose cell tumor (2)

Theca-granulosa cell tumor (1)

In this review of 438 girls examined between 1988-1998, prior to the newer PROS definitions, the incidence of central precocious puberty (CPP) was noted to be 97.7% and GnRH independent precocious puberty (GIPP) was 2.3%. (24) Neurogenic abnormalities were noted in 18.4%, and idiopathic CPP in 74% of the girls in this study. The frequency of neurogenic CPP tended to be higher in the youngest girls (i.e., those under age 4 years) and the frequency of idiopathic CPP tended to be higher in girls presenting at older ages (i.e., between ages 7-7.9). Those girls identified with idiopathic precocious puberty after age 7 may, in fact, represent the recent observations of earlier onset of normal puberty by Herman-Giddens. (3)

Central Precocious Puberty

Central precocious puberty results from early maturation of the hypothalamic- pituitary-gonadal axis. Serum gonadotropins, gonadal pulsatility and sex steroid concentrations are in the normal postpubertal range. As mentioned previously, idiopathic precocious puberty seems to be the most common cause of CPP. Neurogenic CPP seems to be found more frequently in extremely young girls with the earliest onset of puberty. CNS lesions identified include neoplasms, trauma, hydrocephalus, post infectious encephalitis, congenital brain defects, and such genetic disorders as neurofibromatosis type 1 and tuberous sclerosis, and granulomas of tuberculous origin. The most commonly identified neurogenic neoplasms found in CPP include hamartomas, astrocytomas, and pituitary microadenomas. (24) Hamartomas are congenital hypothalamic malformations that histologically contain fiber bundles, glial cells, and GnRH- secreting neurons and often act as a mini-hypothalamus. Less frequently identified tumors include epipendymomas, gliomas, and pinealomas. While the craniopharyngioma has usually been associated with delayed puberty, it can rarely cause precocity as well.

Known genetic causes of CPP are rare and are currently limited to the KISS1 and the MKRN3 genes. The former gene produces a peptide that many currently believe to be the primary stimulatory signal of puberty and the later gene seems to be related to an inhibitor of GnRH secretion that exists prior to puberty, Activating mutations have been found in the genes encoding kisspeptin 1 (KISS1) and its receptor (KISS1Rr1.(34-37) In addition, as in normal puberty, higher levels of kisspeptin 1 have been identified in children with CPP compared to controls.(38) Recent research on 15 families with central precocious puberty utilizing whole exome sequencing identified loss of function mutations in MKRN3 genes which encode the makorin RING-finger protein 3 in 5 of the 15 families. The gene is maternally imprinted and likely plays a vital role in developing cells, particularly in the central nervous system. Interestingly, a larger deletion of 15q11-q13 which contributes to Prader-Willi syndrome encompasses the MKRN3 gene. The protein, makorin RING finger protein 3, is involved with RNA binding and ubiquination and degradation. Further research in 215 unrelated children with sporadic CPP identified 8 children with mutations in MKRN3, all on the paternal allele. (39) While these mutations are rarely a cause of CPP, this research does suggest an inhibitory role of MKRN3 in GnRH secretion. (40)

Other chromosomal abnormalities associated with CPP have also been described, such as 9p deletion, Williams-Beuren syndrome (1q11.23 microdeletion), and 1p36 deletion. Also, maternal uniparental disomy of chromosome 14 (Temple syndrome) and 7 (Silver-Russell syndrome) have been identified. The latter two genomic imprinting disorders, taken into consideration with MKRN3 mutations and Prader Willi, suggest the importance of epigenetic alterations in the pathogenesis of precocious puberty. (41)

Girls with severe primary hypothyroidism can develop true precocious puberty. These girls have elevated gonadotropins in addition to high TSH levels. The associated precocity may result from cross-activation of the FSH receptor by the high circulating TSH or from direct stimulation of the ovary by the gonadotropins. Large ovarian cysts are not uncommon in patients with primary hypothyroidism and precocious puberty. These girls will have the atypical finding for precocious puberty of delayed bone maturation.

Occasionally, treatment and correction of long standing virilizing congenital adrenal hyperplasia will be followed by the development of true precocious puberty. It has been hypothesized that GnRH secretion and gonadotropin stimulation of the ovary may ensue in these patients after the removal of hypothalamic androgenic suppression.

Contemporary Issues for Management of CPP

The evaluation of true precocious puberty requires confirmation of true puberty, a careful physical examination with attention to growth charts, and evaluation for a central lesion. If a CNS lesion is present, the child will typically have a pubertal gonadotropin response to GnRH that is usually associated with idiopathic true precocious puberty and occasionally with a hamartoma. The mainstay of CNS evaluation is imaging of the CNS.

In addition, bone age X-rays are helpful to identify the advanced physiologic age associated with true precocious puberty. Precocious development that continues to progress is almost always associated with a marked increase in growth velocity and sometimes this rapid growth occurs prior to the presentation of precocious development (42).

The long standing gold standard in the diagnosis of central precocious puberty has been the GnRH stimulation test. Peak levels of LH greater than 3 - 5 mIU/ml 30 – 40 minutes following stimulation are highly suggestive of central precocious puberty. (43) After GnRH was no longer available, in the United States, a GnRH-agonist was substituted as the stimulus. Either test remains today the gold standard for diagnosis. The measurement of a single LH value 30 - 60 minutes after administration of a GnRH agonist (leuprolide acetate at 20 mcg/hg) was considered adequate for diagnosing CPP; an LH value greater than 9.2 mIU/ml at 30 minutes was diagnostic in one study. (43,44). Today, however, with the use of ultrasensitive LH assays, it has become standard to use basal LH serum levels as the routine for diagnosis, saving the gold standard stimulation test for those patients with inconclusive unstimulated basal results. (39) Generally speaking, LH values are unmeasurable before pulsatile GnRH is secreted in the prepubertal period. Random LH levels greater than or equal to 0.3 mIU/ml were 100% specific in one study for distinguishing CPP. (45) An unstimulated LH value of 1.1 IU/L or greater has been considered sufficient to assume that endogenous GnRH is being secreted and diagnostic for CPP in another study. (46,47) One should remember that exclusion of central precocious puberty does not rule out gonadotropin independent puberty.

Ovarian imaging and thyroid testing may also complement the evaluation. Estradiol levels are not really helpful in the diagnosis of precocious puberty with one exception. Levels vary tremendously and estradiol levels may be in age appropriate normal ranges in girls with central precocious puberty. If, however, levels are markedly elevated (above 100 pg/ml) then it is likely that the patient either has an ovarian cyst or an ovarian steroid producing tumor such as a granulosa cell tumor.

While some CNS lesions will need treatment (often surgery), the majority of remaining causes of true precocious puberty (i.e., idiopathic) respond to GnRH analogues. It has also been demonstrated that precocity associated with hamartomas, which may intrinsically produce GnRH, may be effectively treated with GnRH agonists. (48) Analogues work by desensitizing the pituitary and decreasing the release of luteinizing hormone and follicle stimulating hormone. (49)

GnRH agonist therapy initially increases circulating gonadotropin and estradiol concentrations for short periods of time. Chronic therapy is associated with suppression of pulsatile gonadotropin secretion and a blockade to the LH response of endogenous GnRH. Suppression is best monitored with GnRH challenge tests although basal LH values may be substituted when there is no doubt about suppression. Some children who are initially suppressed will escape suppression and require increased dosages. Additionally, measurement of serum estradiol (if elevated on prior analysis), height, bone age, and assessment of secondary sexual characteristics may be helpful. Evaluation of ovarian morphology and uterine size by pelvic ultrasonography may, in some cases, provide additional evidence of such suppression.

Cessation of menses, regression in physical pubertal signs (i.e., breast size and pubic hair), and a diminution of uterine and ovarian size usually occur within the first 6 months of therapy. (50) Optimal time for discontinuation of treatment has not been established, however, discontinuation at age 11 appears to result in optimal height outcomes.(51) Pubertal changes reappear within months after cessation of therapy with a mean time to menarche of 16 months.(52)

Analogues can be given in Depot formulations (IM or SC injections q4-12 weeks), as an implant (q4week to 12 month) or as a nasal spray (1-3 times daily). Leuprolide intramuscular injection is the only available depot preparation in the United States, and no studies have documented greater adherence to the multi-monthly dose compared to monthly dose. Injection site reactions occur in 10-15% of patients.(53) Histrelin, the once yearly subcutaneous implant, can suppress gonadotropin secretion for up to 2 years. (54) A minor surgical procedure is necessary for implantation and some site reactions have been reported, even a risk of infection. A recently published open label phase 3 multicenter histrelin study documented the efficacy in sustained gonadotropin suppression with yearly histrelin implants for up to 6 years of use. 52.8% of participants experienced site reactions, all of which were mild to moderate in sequelae. Additional difficulties with implant breakage (22%) at removal were noted. Gonadotropin levels returned to puberty levels within 6 months of implant removal.(50)

The literature does not include randomized controlled trials of long term outcomes for children with central precocious puberty treated by GnRH analogues. Predicted height has been shown to often improve after long-term GnRH agonist therapy; the absence of treatment has been associated with reductions of these height predictions (51,55). In one large study mean gains ranged from 3-10 cm in girls treated up to age 11 years after treatment with GnRH therapy (56). In comparison, one small study of children followed for 12 years with slowly progressive precocious puberty did not demonstrate a loss of adult height without treatment. However, these studies often have flaws such as the calculations of gained height based on unreliable predicted heights.

A consensus document of 30 experts from Europe, the US, and Canada concluded that: “The efficacy of GnRH analogs in increasing adult height is undisputed in early-onset (i.e., girls under age 6 years) precocious puberty” (57). Those children who do not benefit may have the following characteristics: slowly progressive puberty, the precocity of which does not adversely affect the child; a normal predicted height prognosis; and a lack of evidence for gonadal activation (58). While consideration should be given to withholding treatment for these children, studies consistently demonstrate that girls presenting under age 6 years are able to subsequently achieve normal adult height because of the GnRH agonist therapy (59,60). Two of the most difficult decisions in the treatment of central precocious puberty are whether to initiate treatment in girls between ages 6-8 years and to decide what age to stop treatment (61).

Since GnRH agonists decrease the aberrantly increased GH secretion seen in precocious puberty, some have suggested that these analogues may significantly suppress growth velocity enough to compromise the predicted improvement in height which could explain the ambiguity in studies regarding analogue impact on adult height. Some studies have evaluated the benefit of GnRH agonists with growth hormone (GH) and a recent meta-analysis suggested greater final height and predicted adult height with combination therapy, but no difference in final height standard deviation scores. (62) A prospective cohort study evaluated GnRH agonist alone (n= 17) vs GnRH agonist and GH (n=23) and followed subjects until final adult height was achieved. Final adult height was significantly greater than target adult height in the combination treatment group (4.86 +/-0.9cm vs 1.51 +/- 1.0cm, p<0.05) suggesting benefit to the addition of growth hormone to GnRH analogues in CPP. (63)

The psychological effects of central precocious puberty have not been adequately studied (57). Therefore, decisions regarding whether and when to initiate treatment or stop treatment based on psychosexual concerns rely on clinical expertise and expert opinion.

Incomplete, Isosexual, or Gonadotropic Independent Precocious Puberty (GIPP)

GIPP can originate from the gonads, the adrenals, from extragonadal or intragonadal sources of human chorionic gonadotropin, or from exogenous sources. In girls, functionally autonomous ovarian cysts are the most common cause of GIPP. An ovarian follicle up to 8 mm in diameter are common in normal prepubertal girls and may appear or regress spontaneously, but rarely secretes significant amounts of estrogen (64,65). An intriguing finding of the somatic cell mutation associated with McCune-Albright syndrome in the cells of one such cyst sheds light on this occurrence (66). GnRH agonists are not effective in treating autonomous cysts.

Juvenile granulosa cell tumors or theca cell tumors of the ovary are a rare cause of GIPP. Tumor markers for granulosa cell tumors include Inhibin B and müllerian inhibiting substance. Other ovarian neoplasms even more rarely seen in this age group that may also secrete either estrogens and/or androgens include gonadoblastomas, lipoid tumors, cystadenomas, and ovarian carcinomas (67). Peutz-Jeghers syndrome has been associated with GIPP; the mucocutaneous pigmentation and gastrointestinal polyposis seen in this disorder has been rarely associated with gonadal sex-cord tumors (68).

McCune-Albright syndrome (MAS) classically includes the triad of hyperpigmented café-au-lait spots, progressive polyostc fibrous dysplasia of the bones and GnRH-independent sexual precocity (69). Some girls will present with vaginal bleeding preceding thelarche. Bone lesions and café-au-lait spots may increase over time. The actual clinical phenotypes vary markedly.

This disorder is caused by postzygotic somatic cell mutations of the gene encoding the alpha-subunit of the stimulatory guanine nucleotide binding protein Gs. These activating mutations stimulate constitutive G protein activation in affected cells with aberrant cyclic AMP production (70). The mutations may occur at various times in fetal development with a patchy tissue distribution of affected cells. Each of the associated findings is affected by these mutations: granulosa cells in the ovary, melanocytes of the skin (71), and the dysplastic bone cells (72,73). In addition to the classic triad, other endocrine cells may also be similarly affected and associated with their autonomous hyperfunction: pituitary adenomas, usually growth hormone secreting, hyperthyroid goiters (74), and rarely adrenal hyperplasia (75). Another recent finding is the presence of these same somatic cell mutations in cells from isolated hyperfunctioning ovarian cysts of GIPP patients who do not exhibit other findings of McCune-Albright Syndrome (66). This may account for the findings of “ovarian hyperfunction” in patients with GIPP as reported in the series of Table II above (24).

The sexual precocity of McCune-Albright syndrome is due to autonomously functioning follicular cysts. These patients can progress from GnRH independent to GnRH dependent puberty; when their bone age reaches the physiologic age of the normal time-onset for puberty, awakening of the arcuate nucleus for pulsatile GnRH secretion may occur and progress to the establishment of ovulatory cycles.

Approaches to treatment have included aromatase inhibitors such as Testolactone and selective estrogen-receptor modulators. Studies evaluating the efficacy have been uncontrolled. One study with Testolactone showed only early effectiveness, with loss of efficacy over time (76). Another study showed success with Tamoxifen in reducing vaginal bleeding (77). However the effect of Tamoxifen on height has not been adequately evaluated. One international multicenter trial evaluated the efficacy of monthly fulvesterant in 30 girls with MAS and followed them for a year. Days of vaginal bleeding, and bone advancement were less in the treatment patients. However, there were no changes in predicted adult height or frequency of ovarian cysts.(78) An open label study evaluating the effectiveness of letrazole on 9 girls with MAS for 12-36 months demonstrated reduction in rates of growth, vaginal bleeding and bone age. Ovarian volume, estradiol, and bone metabolism indices which showed initial improvement, began to rise after 24-36months of treatment. (79) When the shift from gonadotropin independent to gonadotropin dependent puberty takes place, GnRH analog therapy then becomes the first line therapy.

Iatrogenic sexual precocity

In prepubertal children, exogenous intake of estrogen has been shown to cause precocious pubertal development. Estrogen containing products may include variety of health or nutritional supplements and personal products such as hair products, lotions, and creams. Ingestion of estrogen containing meat has also been implicated although controversial. In actuality, these causes of precocious development appear to be extremely rare.

Premature Thelarche

Isolated precocious thelarche is a common entity and is associated with unilateral or bilateral breast enlargement without other signs of sexual maturation. It generally occurs at early ages up to 4 years, with approximately 80% presenting prior to age 2 years. The thelarche regresses spontaneously after diagnosis in over half of girls (80).

In all girls gonadotropin levels rise in the newborns after delivery and remain elevated for up to 4 years of age. While most newborns rarely exhibit a dramatic ovarian response to these elevated levels, it is likely that isolated precocious thelarche is a result of this physiologic process. The uterus remains prepubertal in size during this time, however, the ovaries may develop temporary follicular activity, and estradiol levels will be slightly higher than is seen in control girls. This is usually a benign self-limiting disorder not associated with bone age progression. However, clinical consideration should be given that the breast development could be the first sign of precocious puberty. A careful history and physical assessing for neurological symptoms and signs and assessment for growth by growth charts and a bone age should usually be performed.

Premature Menarche

Premature menarche has been reported as periodic vaginal bleeding without other signs of secondary sexual development (81). While this entity has been repeatedly yet rarely reported, pediatric vaginal bleeding can occur as the first manifestation of sexual precocity in most causes of GIPP listed above. These etiologies should be excluded before one considers premature menarche as the diagnosis. The differential diagnosis of vaginal bleeding in a child without other signs of sexual maturation is quite different than precocious development and includes foreign objects in the vagina (common) and vaginal tumors (rare).

Contrasexual precocity

Virilizing precocious puberty in girls and isolated precocious adrenarche

Most girls with contrasexual precocious puberty present with early appearance of pubic hair or hirsutism. The most common cause is a mild form of 21-hydroxylase deficiency, which is present in 0.1-1.0% of the population. Other more rare forms of congenital adrenal hyperplasia have also been identified in these patients. Virilizing adrenal (occasionally malignant) and ovarian tumors (e.g., Leydig or Sertoli cell tumors) in young girls can similarly present with virilizing precocious puberty. In actuality, most girls with appearance of pubic hair likely have isolated precocious adrenarche. While many of them have only early yet normal pubertal development (3), evidence exists that the prevalence of ovarian hyperandrogenism, hyperinsulinism and dyslipidemia is increased in this population (82). These findings suggest that premature pubarche in some girls may be a childhood marker for insulin resistance and polycystic ovary syndrome.

DELAYED PUBERTY

An Overview of Delays within the H-P-O Circuit (Delays of Secondary Sexual Development and Menarche)

Several large descriptive studies have been published which have categorized the causes of pubertal/ menarchal delay. In 1981, a series of 252 female adolescents evaluated over 20 years at the Medical College of Georgia from a large referral area in Georgia was published (83). It included all patients seen with either delay of the onset of puberty or menarchal delay. The series was subsequently expanded to include 326 patients. In this series the most common causes of abnormal puberty were: (1) ovarian failure (now called ovarian insufficiency) (42%); (2) congenital absence of the uterus and vagina (14%), and (3) constitutional delay of puberty (10%). While these 3 disorders comprised two-thirds of all patients seen, a host of less frequent disorders was also diagnosed (see Table III below); the most common of these included PCOD and idiopathic hypogonadotropic hypogonadism (IHH), both at 7% each.

Table III. Etiologic breakdown of 326 patients with abnormal puberty (pubertal and menarchal delay) (Medical College of Georgia Series) (84)
	Group total	No.	%
Hypogonadism (Pubertal Delay) Hypergonadotropic hypogonadism:
Turner Syndrome	84		26
Chromosomally Normal	57		16
46,XX		48	15
46,XY		9	2
Total	141	57	43
Hypo (eu) gonadotropic hypogonadism:
Reversible	62	18
Constitutional delay		32	10
Systemic illness		7	2
Eating disorders		9	3
Primary hypothyroidism		4	1
CAH		3	1
Cushing syndrome		1	0.5
Pseudopseudohypoparathyroidism		1	0.5
Hyperprolactinemia		5	1.5
Irreversible	37		13
Congenital Deficiency Syndromes
Isolated GnRH deficiency		23	7
Forms of hypopituitarism		6	2
Congenital CNS defects		2	0.5
Acquired anatomic lesions
Unclassified pituitary adenoma		2	0.5
Craniopharyngioma		3	1
Unclassified malignant tumor		1	0.5
Total	99		31
Eugonadism: (Menarchal Delay)
Anatomic	59	18
Mullerian aplasia		45	14
Outlet obstruction
Transverse vaginal septum		10	3
Imperforate hymen		2	0.5
Cervical atresia		1	0.5
Inappropriate feedback	22		7
Intersex disorders	5		1.5
Androgen insensitivity		4	1
17-ketoreductase deficiency		1	0.5
Total	86		26

In April of 2002, a more recent series of both male and female patients evaluated for delayed puberty at Children’s Hospital in Boston between January 1996 and July 1999 was published (85). This study, like the MCG study, included patients with delayed onset of puberty; it, however, did not include patients with menarchal delay. For the females reported (N=74), the 3 most common causes were: (1) constitutional delay of puberty (30%); (2) ovarian failure now called ovarian insufficiency (26%); and permanent hypogonadotropic hypogonadism (20%). Over 20 other numerically less frequently reported disorders were identified and listed below (see Table IV).

Table IV. Etiologic breakdown of 74 females with delayed puberty (Children’s Hospital Series, 2002) Revised from Sedlmeyer, et al. (85).
	Group total	No.	%
Hypogonadism (Pubertal Delay) Hypergonadotropic hypogonadism:
Turner Syndrome	5		7
Chromosomally Normal	14		19
46,XX		13	17
46,XY		1	2
Total	19	14	26
Hypo (eu) gonadotropic hypogonadism:
Reversible (Functional)
Constitutional delay		22	10
Systemic illnes		1
Giardiasis		1
Rheumatoid Arthritis		1
Systemic lupus erythematosis		1
Sickle cell disease		1
Congenital heart disease		1
Isolated seizure disorder		1
Eating disorders
Endocrine disorders		2
Growth hormone deficiency		1
Hyperprolactinemia		1
Irreversible (Permanent)		15	20
Congenital/ Genetic Syndromes
Kallmann syndrome		1
Idiopathic Hypo Hypo		2
CHARGE syndrome		2
Forms of hypopituitarism
Rathke's pouch		2
Hypophysitis		1
Hypopituitarism		1
Panhypopituitarism with hearing loss		1
Acquired anatomic lesions
Craniopharyngioma		3
Germinoma		1
Ologodenrdroglioma		1
Total	51		67
Other	4		5

Numerical and physical clues to the disorders presenting with delays in pubertal development: organizing the approach to the patient.

The numerical findings in these series point out several useful facts. First, most practitioners confronted with females presenting with pubertal delay can identify a few disorders that present in the majority of patients: ovarian insufficiency, constitutional delay, and permanent hypogonadotropic hypogonadism (as frequent causes of delayed onset of puberty) and vaginal agenesis (as the most frequent cause of menarchal delay). Rather than wait until the ages defining female pubertal or menarchal delay (ages 13 and 15 or 16 years, respectively), a physical examination with inspection of the introitus, plotting the patients on growth charts (longitudinal and velocity), and obtaining gonadotropins values will identify many of these disorders even before these age definitions are met. Idiopathic hypogonadotropic hypogonadism (IHH), however, is the exception being more difficult to diagnose in the younger patients. It is often a diagnosis of exclusion in the late teenage years. Secondly, constitutional delay occurs in less than one-third of patients in any series. While constitutional delay is a frequent cause of delayed puberty it occurs with higher frequency in males, and less frequently in females. Two thirds of all females presenting with delayed puberty historically have had underlying pathology. Lastly, pubertal delay can be an ascertainment for the identification of a rare disorder (See Table II). Similarly, should any diagnosis be made during childhood years and in advance of the time for normal puberty, plans can be made prior to the pubertal years to begin treatment and to allow for the most normal pubertal progression as is possible. At least in the Children’s Hospital setting, this appears to be the case for Turner syndrome for which the frequency of presentation with delayed puberty was decreased from the earlier MCG series.

The physical findings of the patients in these series also provide clues for helping us to form a differential diagnosis and organize our diagnostic approach. First, classification according to estrogen as in the MCG series allows for a separation of major etiologies.

Table V. Classification of Pubertal Abnormalities

I. Hypoestrogenism/ Hypogonadism (Delayed Onset of Puberty)

A. Ovarian failure (Hypergonadotropic)

B. Hypothalamic-Pituitary Immaturity or Suppression (Hypogonadotropic)

II. Normal estrogen milieu/ Eugonadism (Delayed Menarche)

A. Congenital absence of uterus and vagina (CAUV)

B. Chronic Anovulation (e.g., PCOD)

C. Intersex Disorders (e.g., Androgen Insensitivity)

The absence of breast development suggests a cause of hypogonadism: ovarian failure / ovarian insufficiency or a hypothalamic-pituitary problem. The practitioner can further narrow these possible etiologies by obtaining an FSH level; high levels suggest ovarian insufficiency and low normal values direct one to etiologies that have their effect at the level of the hypothalamus or pituitary. The presence of breast development usually directs one towards causes of menarchal delay suggesting the ongoing production of estrogen. One should remember, however, that some patients may have initiated puberty only then to have this process (and estrogen production) suppressed. Historically, biological evidence for estrogen or its lack has been more helpful than a single estradiol assay. A vaginal smear which demonstrates greater than 15% superficial cells, a positive progestin challenge test, the presence of endometrium on ultrasound measuring 1.5 mm or greater, or the presence of copious cervical mucus will usually confirm the suspicion of ongoing estrogen production.(86) There are currently no available studies for which evidence supports or refutes any one best method of determining the presence of sufficient ongoing estrogen production. Patients demonstrating breast development in the absence of evidence of ongoing estrogen production by any of these methods should be treated like any other hypogonadal patient.

Second, absence of pubic hair after age 13 years is a very significant clue of several specific abnormalities. Pubic hair growth results from both adrenal and gonadal androgen production. One should remember that even when the H-P-O circuit appears delayed, the H-P-A (adrenal) circuit should still be functioning and providing adrenal androgens. For most disorders of delayed onset of puberty, at least some pubic hair should be present because this H-P-A circuit is unaffected by the defect (ovarian insufficiency and IHH). When pubic hair is absent after 13 years, it suggests a defect of: (1) pituitary function (i.e., the inability to stimulate both ovarian and adrenal androgen production as in pituitary insufficiency); (2) steroidogenesis (i.e., the inability to convert cholesterol to androgens as in 17-hydroxylase deficiency); or (3) androgen receptors (i.e., the inability to translate the hormone signal into end organ androgenization as in Androgen Insensitivity Syndrome (AIS)). The first two of these disorders occur in the 46,XX hypogonadal patients (Tables III and IV) and demonstrate defects within both H-P-O and H-P-A circuits, the common denominator being pituitary insufficiency or a steroid enzyme block. When examined they are found to have a normal müllerian system. 46,XY patients with 17-hydroxylase deficiency will present with absence of: (1) pubic hair; (2) breast development; and (3) a müllerian system. Androgen receptor defects are found in patients with normal breast development and absence of the vagina (i.e., AIS). Thus, for the patient with absent pubic hair after age 13 years, the most critical portions of the examination include the breasts and introitus.

Third, the apparent absence of a müllerian system (i.e., vaginal agenesis) can occur for either 46,XX or 46,XY patients. However, an examination, not a karyotype, is the most cost effective initial screen. Patients may present with absence of the vagina yet also demonstrate normal pubertal breast and pubic hair development. If a rectal examination is unrevealing for them, the likely diagnosis is congenital absence of the uterus and vagina (CAUV) also known as müllerian aplasia or Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. If, instead, a bulging midline mass is identified just above the “absent vagina,” the patient likely has either a transverse vaginal septum (TVS) or imperforate hymen. None of these findings warrant chromosomal studies as they clinically suggest the presence of a 46,XX karyotype. The patient found to have breast development and absence of both pubic hair and a müllerian system likely has AIS. These latter findings alone warrant a karyotype to confirm the 46,XY compliment and the need for gondadal extirpation. As stated above, the patient with absence of the müllerian system as well as thelarche and adrenarche likely has 46,XY 17-hydroxylase deficiency.

Fourth, identification of stature significantly shorter than one would expect for an individual whose growth was interrupted only by the delayed onset of puberty often reveals a genetic cause for both short stature and delayed puberty (e.g., Turner syndrome). Alternatively these findings could be the result of an endocrine cause which stopped growth several years earlier than the usual time onset for puberty in addition to preventing or slowing the onset of secondary sexual development (i.e., growth hormone deficiency, thyroid deficiency, or pituitary insufficiency).

DISORDERS IDENTIFIED IN PATIENTS WITH EITHER DELAYED PUBERTY OR MENARCHE

The remainder of this chapter will address specific concerns of the most common causes of the pubertal abnormalities identified in the two series described above. It will primarily refer to the data of the MCG updated series of 326 patients presenting with either delayed pubertal onset or delayed menarche tabulated in Table III and classified according to Table V above (84). In addition to discussing the common findings associated with these etiologies it will point out recent findings from molecular medicine and summarize contemporary treatment strategies.

Hypogonadism

Hypergonadotropic Hypogonadism

The single most common cause of delayed puberty in all prior delayed puberty series has been primary ovarian insufficiency (83,84). Forty-three percent of all patients seen in the MCG series had hypergonadotropic hypogonadism. The fact that ovarian insufficiency presenting at puberty was numerically less frequent (i.e., 26%) in the recent Children’s Hospital series suggests that more children are being diagnosed with Turner syndrome and other forms of ovarian insufficiency before the adolescent years and that treatment may be presently initiated at an earlier age (85). In future series of delayed puberty, primary ovarian insufficiency may all but disappear as an etiology; ideally these patients being diagnosed before the usual time onset of puberty with earlier initiation of treatment.

Turner Syndrome

Numerically, more patients with ovarian insufficiency and delayed puberty have had a form of Turner syndrome than were diagnosed with either 46,XX or 46,XY gonadal dysgenesis. Approximately 30% of the Turner patients have the classic 45,X karyotype with the remainder of patients having mosaic forms of Turner syndrome (Table VI below). Mosaicism refers to the presence of two or more cell lines, both of which originated from a single cell line. Patients with mosaic forms of Turner syndrome usually have a 45,X cell line associated with another cell line such as 46,XX or 46,XY. Other cell lines exist which represent structural abnormalities of the X chromosome such as isochromosome for the long arm of X, i.e., [i(Xq)] ; they may occur either as single cell lines or as mosaicism in association with 45,X.

Table VI. Karyotypes of patients with CIOF. Reproduced with permission (83)
Classical Turner Syndrome (45,X)		28*
Y Cell Lines		16
46,XY	1*
45,X/46,XY	12
45,X/47,XY	1
45,X/46,X?del(Y)	1
45,X/46,X,i dic(Y)/47,XY,i dic(Y)/ 46,XY/47,XYY	1
Structural abnormalities of X		31
Isochromosome
46,X,i(Xq)	7*
45.X/46,X,i(Xq)	10
45,X/46,X,i dic(Xq)	2
45,X/46,X,i (Xq)/46,i (Xq),i (Xq)	1
45,X/46,X,i (Xq)/47,X,i (Xq),i (Xq)	2
Other
46,X,t (X;X)qter-p22	1*
45,X/46,X,del X (q13)	2
46,X,Xq+	1*
45,X/46,X,Xq+	1
45,X/46,X,r(X)	1
45,X/46,XX/46,X,r (X)/ 47,X,r (X),R (X)	1
45,X/46,X,r	1
46,X,del X (q25)	1*
Other X mosaic cell lines		9
45, X/46, XX	8
45,X/47,XXX	1
Total	84
* Single cell lines.+ Turner phenotype with intra-abdominal streak gonad and contra-lateral intra-abdominal testis.

All of the chromosomal findings in mosaic and non-mosaic patients with Turner syndrome have a common denominator: privation of either the entire X chromosome or a portion of the X chromosome. Fetuses with Turner syndrome have as many germ cells at mid gestation as do 46,XX fetuses. It is commonly believed that the loss of critical X chromosome-linked ovarian determinant gene(s) (87-89) is the cause of accelerated loss of germ cells (90) due to a defect of follicular development as noted by Jirasek et al. Many of these individuals lose all of their follicles with associated germ cells before birth. Some of them lose the remaining germ cells during childhood years and before puberty. Less than 15% of patients with Turner syndrome will lose their follicles (with germ cells) either during or after the pubertal process (83). Five percent of patients with Turner syndrome will have enough follicles (i.e., germ cells and surrounding granulosa cells) remaining at puberty to not only initiate the pubertal process but also to allow them to have regular, cyclic menses during at least a portion of their adolescent or adult years; 2-5% may spontaneously become pregnant.(91,92)

Once the germ cells are prematurely depleted from the ovaries, the only remaining tissue present is the connective stroma of the gonads. It usually appears as a ribbon of white connective tissue located beneath the fallopian tubes and along the pelvic sidewalls (90). These residual gonads have the appearance of “streaks” and are referred to as streak gonads. The presence of a Y cell line in a patient with Turner syndrome brings with it a 15-25% risk of developing malignant germ cell tumors within those streak gonads. In those particular patients the streaks need to be surgically removed as soon as a diagnosis is made. For all patients with Turner syndrome, privation of X chromosomal material is associated with the variable Turner stigmata, cardiovascular and renal abnormalities, and the development of a number of specific medical problems. Turner stigmata include short stature, high arched palate, low hair line and webbed neck, multiple pigmented nevi, short fourth metacarpals, shield chest, increased carrying angle of the arms (cubitis valgus), and lymphadema of ankles, to name a few.

These stigmata related to loss of X-chromosomal material are variably present in Turner patients. Furthermore, reports of phenotypic-karyotypic correlations have been inconsistent (83,93). Several observations and hypotheses have been made that help understand these relationships or lack thereof. First, it has often been felt that the presence of physical findings associated with Turner syndrome is dose dependent, i.e., the higher the percentage of 45,X cells the greater the likelihood of such abnormalities. While this makes the greatest sense intuitively, not all studies have been able to demonstrate a relationship between karyotype and phenotype (83). Recently, when ascertainment was considered, better correlations were made dependent on the degree of mosaicism. Patients found incidentally by prenatal karyotyping had fewer phenotypic features of Turner syndrome than those diagnosed after birth because of a clinical suspicion (94). Another explanation suggests that X chromosome gene imprinting exists and that some of the findings of Turner syndrome are related to the parental origin of the missing X chromosome in Turner patients (95).

Short stature is the one consistent phenotypic finding of Turner syndrome (83). The MCG series was reported prior to the treatment of Turner patients with growth hormone. The fact that none of the patients in that series was taller than 63 inches (160 cm) in height supported the tenet that statural genes are located on both arms of the X chromosome. The knowledge of consistent short adult stature, often under 5 feet (152 cm), and the potential psychological effect it has in combination with other features of Turner syndrome, provided impetus for identifying therapies independent from estrogen treatment for these patients. Many hundreds of Turner patients have now been treated with growth hormone pushing the final adult stature beyond this 63-inch (160 cm) mark for some and certainly past the predicted final height for many other Turner women.

The most serious somatic abnormalities found in patients with Turner syndrome are those involving the heart and great vessels. Cardiovascular disease is the primary cause of early mortality in women with TS with standard mortality ratios of 3.5 (CVD) to 24 (congenital anomalies).(96) Most of the mortality results from cardiac malformations, which have been reported in up to 50% of patients and include coarctation, pseudocoarctation, bicuspid aortic valves (separately between 30 and 45% incidence), and a host of other anatomic variants of the vascular tree, especially in the area of the ascending aorta. The high prevalence of these abnormalities has been reported in the years following the NIH consensus panel as has the recommendations for routine MRI screening (97-99). 1.4% of Turner patients have been estimated to develop dilation of the ascending aorta with subsequent dissection and rupture; most have died after being misdiagnosed with another cause of the chest or epigastric pain (97-101). Most patients with dissection and rupture of the ascending aorta have had a cardiac congenital malformation, hypertension, or pre-existing dilation. At least 10%, however, have had neither an identifiable risk factor including aortic dilation nor an aorta diameter above the previously held risk size (i.e., > 5 cm) (101). Several explanations have been given for dissection and rupture in patients not felt to be at risk. First, this occurrence has been associated with the pathohistologic entity of cystic medical necrosis of the vessel wall, the culprit of similar clinical outcomes in patients with Marfan syndrome. This suggests that there is an inherent defect of the vessel wall that predisposes all Turner women, with or without risk factors, for this occurrence (100). Second, prior measurements have not taken into account the fact that women with Turner syndrome are smaller and thus should have smaller size aortas. When the aorta size was normalized to body surface area in a study of 166 adult Turner patients and compared to a control population (n=26), over 30% of the Turner women had an ascending aorta measurement that was larger than that of 95% of the control population (99,101). As a result, new guidelines have been suggested for those aorta measurements above which significant risk for rupture exists (99,101,102).

Pregnancy may be the largest single risk factor for dissection and rupture of the aorta in Turner patients. There are nearly a dozen reports in the literature of death occurring during, immediately after or even more remotely removed from pregnancy in Turner patients who became pregnant from oocyte donation and embryo transfer. This gathering body of literature supports the fact that the cardiovascular (i.e., increased blood volume and stroke volume) and potential hormonal changes of pregnancy (perhaps remodeling of vessel wall by estrogen or progesterone) place these patients at a high risk of dissection, rupture of the ascending aorta, and death (101,103-105). A conservative estimate of a 2% maternal mortality rate has been reported from a US national survey and is 100 fold greater than the death rate for all causes during pregnancy (103). Similarly, a French study demonstrated a maternal mortality of 2.2%.(106) While death usually occurs during pregnancy, some evidence suggests that changes of the aorta during pregnancy may increase the risk of rupture in future years as well. The report of a more recent Nordic cohort study of pregnant Turner women did not find maternal deaths but did report: 35% hypertensive disorders; 20% of patients with pre-eclampsia; and a 3.3% potentially life threatening problem, (107) Further prospective longitudinal data are needed to understand the absolute risk to these women during pregnancy. It would be ideal if IVF registries included this information.

A number of other medical conditions may also be found in Turner patients. Horseshoe kidney is the most common renal abnormality observed and a number of autoimmune disorders, commonly Hashimoto thyroiditis, are diagnosed. Given the higher incidence of specific medical conditions for women with Turner syndrome than the general population, the NIH study group guidelines recommend continued monitoring of hearing and thyroid function, screening for hypertension, diabetes, and dyslipidemia as well as aortic enlargement (98).

Normal Chromosomes

The second largest group of young women with primary ovarian insufficiency has a 46,XX karyotype (46,XX gonadal dysgenesis). For them, some have a genetic etiology. An autosomal recessive form of this disorder was previously suggested by the presence of sibships reported in which several non-twin sisters are affected with ovarian insufficiency (83). The reports of mutations in candidate autosomal genes of affected patients provides support for the belief that autosomal etiologies exist for patients with 46,XX gonadal dysgenesis and premature ovarian insufficiency (POI). However, the more consistent finding that approximately 2% of sporadic and 14% of familial cases of 46,XX ovarian insufficiency have premutations for the fragile X syndrome makes this the current most likely explanation for the presence of 2 or more sisters with ovarian insufficiency (108). In addition, a number of other known genetic disorders have also been associated with POI including myotonia dystrophica, ataxia telangectesia, galactosemia, blepharophimosis-ptosis-epicanthus inversus syndrome, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome, and proximal symphalangism. In addition, infiltrative diseases such as mucopolysaccharidoses and environmental etiologies such as childhood viral illnesses may also cause premature depletion of oocytes from the ovaries. This is suspected in identical twins reported to be discordant for ovarian insufficiency (83). While mumps can cause orchitis in males, it is suspected that viruses such as mumps may cause oophoritis and loss of oocytes as well. Patients previously treated for childhood malignancies such as Wilms tumor, may develop germ cell depletion as a result of radiation therapy or chemotherapy (e.g., alkylating agents).

Probably the most common cause of premature primary ovarian insufficiency in women with a 46,XX karyotype is autoimmune. For the group of patients for whom an abnormality is not identified, autoimmune is considered the most likely cause. These patients have an increased risk for developing other autoimmune endocrine abnormalities such as thyroiditis with thyroid dysfunction, hypoparathyroidism, and adrenal insufficiency. In addition, pernicious anemia has been reported in some of these patients. They should be screened on a routine basis for thyroid dysfunction and the other endocrinopathies, if symptomatic. Previous recommendations for patients with 46,XX POI included annual screening with a.m. cortisol levels followed by an ACTH stimulation test in those whose a.m. cortisol levels measured less than 17 – 20 mcg%. Subsequently, given the low prevalence of adrenal insufficiency in these patients, it was suggested that such screening be contemplated only when Addisonian symptoms presented. The NIH has a high referral ascertainment of POI patients with adrenal insufficiency. Studies of these patients have now shown that routine screening for the presence of adrenal steroid or 21-

hydroxylase antibodies is effective to identify patients at-risk for adrenal insufficiency and, once identified, ACTH stimulation testing can follow (109).

As one would suspect, in the absence of an identifiable genetic etiology for depletion of the oocytes, more of the 46,XX gonadal dysgenesis patients present at puberty with residual germ cells after the initial insult than do those with Turner syndrome. In the MCG series of patients, nearly 40% of them had enough follicles at puberty to mount a pubertal response before presenting with amenorrhea and ovarian insufficiency (83). A number of patients with 46,XX gonadal dysgenesis will actually go through the pubertal process and have cyclic menses before developing ovarian insufficiency and amenorrhea in their late teens or 20’s. Some of these patients who spontaneously go through puberty will also have reversal of ovarian insufficiency for indeterminate periods of time and rarely become pregnant during these times of spontaneous menstrual function. It is because of this natural history of POI that includes the reversal of the disease process in some patients that the term previously used for this condition by Fuller Albright, ovarian insufficiency, has been revived by some current authorities (109,110).

It is difficult to understand accurately the numeric breakdown of the different etiologies of ovarian insufficiency in pubertal delay patients. Reports of large series of such patients exhaustively studied to determine cause do not currently exist. There is, however, information regarding the breakdown of different etiologies in a large French cohort (N=357) of ovarian insufficiency patients spanning the ages of 11 to 39 years from which inferences may be made for the younger population (111). In that series, 7.8% of patients with POI had an identifiable genetic cause including chromosomal abnormalities (not Turner syndrome) (2%), FMR1 pre-mutations (2%), molecular alterations of genes thought to be etiologic (i.e., FSHR, GDF9, BMP15) (2%), congenital disorders of glycosylation (0.2%), and autoimmune polyglandular syndrome (APS) type 2 and multiple autoimmune disease (0.8%). In addition, 10% of the patients presented with an autoimmune disorder not identified as genetic. Ovarian insufficiency in the remainder of women was considered idiopathic.

Rare patients present with 46,XY gonadal dysgenesis. These are patients who likely have mutations in a gene controlling testicular morphogenesis such as the SRY gene, often referred to as the master switch for testicular development. While only approximately 15% have SRY mutations, there are now a number of genes both upstream and downstream in expression of SRY for which mutations may alter testicular development. As a result, the germ cells that arrive at the genital ridge will organize in the cortical, rather than medullary region of the undifferentiated gonad. For these classic patients with 46,XY gonadal dysgenesis, however, germ cell loss is complete before birth. Since they never develop testes, they will not produce müllerian inhibiting substance to ablate the developing müllerian system. They will also not produce androgens to allow for masculinization of the external genitalia.

Historically these 46,XY individuals were labeled with Swyer syndrome; at birth they have a normal female phenotype with a normal vagina, uterus and fallopian tubes., i.e., complete 46,XY gonadal dysgenesis or sex reversal (112). At puberty, they do not initiate pubertal development and are found to have elevated gonadotropin levels. They do not have other phenotypic abnormalities like the patients with Turner syndrome. They are often tall because of the presence of a Y chromosome. 46,XY individuals with gonadal dysgenesis have the highest risk for developing germ cell tumors of their streak gonads of any individuals with gonadal dysgenesis and a Y chromosome cell line. The streaks must be removed as soon after diagnosis as is reasonable. Less frequently, partial forms of this syndrome have been found to exist often in association with other systemic anatomic or medical conditions such as polyneuropathy, adrenal insufficiency, and even sudden infant death syndrome (113,114).

Molecular Findings

Turner syndrome. While Turner syndrome is considered to result from haploinsufficiency of critical loci or regions of the X chromosome and a number of putative genes have been identified, a molecular understanding of mechanisms involved is far from understood. A number of the stigmata and malformations of Turner syndrome have been thought to be caused by edema present during development because of an abnormal lymphatic vascular system and thus abnormal lymphatic drainage. As such, the abnormalities are actually deformations. For example, edema of the nail beds causes nail hypoplasia, edema of the neck causes cystic hygromas and webbed neck, and edema of the kidneys prevents them from migrating around the aortic bifurcation and results in horseshoe kidney. The presence of cystic hygromas during fetal life is also associated with coarctation of the aorta; lymphatic drainage back to the heart is sufficiently abnormal during development to cause this cardiac malformation and likely some of the other anatomic variations of the vascular tree that have been found in these patients.

One region of the X chromosome, Xp11.2-p22.1, has been thought to include “Turner syndrome loci”, as a number of associated features including ovarian insufficiency, short stature, high-arched palate, and autoimmune disease have been mapped here (115). Deletions of the X-chromosome linked SHOX gene has explained many of the dysmorphic skeletal features of Turner syndrome including the short stature (11). While not consistently reported, it has generally been thought that the number of phenotypic findings of Turner syndrome are related to the percentage of cells that are 45,X; the implication being that mosaic patients have fewer findings than do those with a single 45,X cell line. As stated above, a recent correlation between some of the findings associated with Turner syndrome suggested an imprinting effect with the variation in phenotype at least partially explained by the parent of origin of the remaining X chromosome. Renal abnormalities, for example, were exclusively found in patients retaining their maternal X chromosome (95).

Prior karyotypic/phenotype correlations have suggested that the proximal regions of both the p and q arms of the X chromosomes are most critical for maintenance of the germ cell compliment (93). However, terminal deletions at the telomeric regions of these arms are also associated with oocyte depletion, although to a lesser degree. Deletion of these regions are more likely to result in POI after some period of ovarian function rather than a complete loss of germ cells evident at the start of the teenage years as is more commonly seen with the proximal deletions.

Early molecular studies of patients with POI and translocations between the X- chromosome and autosomes identified 2 regions of the long arm of the X chromosome within the translocation breakpoints which were felt to harbor important ovarian determinant genes. POF1 (Xq26-q28) (116) contains several candidate genes (HS6ST2, TDPF3, GPC3) (116) and one known to be associated with POI, the Fragile site Mental Retardation 1 (FMR1) gene. POF2 (116,117) (Xq13.3-q22), the human homologue of the Drosophila melanogaster diaphanous gene, contains several candidate genes for which one, (DIAPH2), has been disrupted in POI (118,119). Other loci on the X chromosome have also been identified as important in maintenance of a normal oocyte compliment. Members of the Transforming Growth Factor-β (TGF-β) superfamily proteins are known to have key functions within the oocytes and granulosa cells. Of them, Bone Morphogenetic Protein 15 (BMP15 or GDF9) is produced by a gene (BMP15) mapped to Xp11.2 (120). Mutations within this gene have been associated with POI (121-123). While the list of X-chromosome candidate genes for ovarian determinants is ever growing, 2 genes known to be important in drosophila ovarian development or oogenesis are the DEAD-box 3 (DBX) and the Ubiquitin-Specific Protease 9 (USP9X) genes. Both of these genes, are located within the human Xp11.4, an area known to escape X inactivation. It would appear that a double dosage of all of these genes, especially DBX and USP9X, is required for normal ovarian function. Mutations, interruption, or loss of one of these genes results in premature loss of germ cells from the ovaries. It is possible that mutations within these loci are responsible for ovarian insufficiency in women with intact X chromosomes as they likely are in patients with Turner syndrome. All in all, there appear to be numerous gene loci on both arms of the X-chromosome responsible for ovarian development and function. It is no wonder that all of the Turner variant chromosomes, each with different portions of the X chromosome missing, result in POI.

The most studied of the X-chromosome genes associated with POI is the FMR1 gene. When mutated by a CGG triple nucleotide repeat expansion the result is fragile X syndrome. As in many triple nucleotide repeat disorders, areas of normal repeat sequence may be predisposed to expansion during or before meiosis. Function of the gene is maintained within a given number of these triple repeats but when a certain threshold is reached gene function may be adversely altered. For the fragile X gene (FMR1), a CGG repeat sequence occurs with up to 60 such repeats being normal. Expansion to over 200 such repeats leads to fragile X syndrome; the high level of repeats causing hypermethylation of the promoter and silencing of the gene. Interesting observations were made that female carriers of the premutation of this locus with an unstable intermediary level of repeats (i.e., 60-199), often had POI. Best evidence suggests that this premutation is associated with a 21 fold greater chance of developing POI and that 2% of sporadic and 14% of familial ovarian insufficiency patients harbor this unstable intermediate trinucleotide repeat. Similarly, microdeletions of the FMR2 gene are associated with the same predisposition to POI (124).

46,XX Gonadal Dysgenesis.

The list of genes involved in ovarian development and maintenance of the germ cell compliment has continued to expand as molecular analysis of patients with 46,XX gonadal dysgenesis and POI has revealed etiologic mutations. Some patients have mutations within one of the X-chromosome loci. For others, mutations have been found within autosomal genes, some that are associated with syndromic POI and others with nonsyndromic forms. Additionally, many, but not all POI or gonadal dysgenesis etiologies are associated specifically with the premature loss of germ cells. Examples of known genes for which mutations have been shown to cause syndromic forms of premature loss of germ cells include the Autoimmune Regulator (AIRE) gene causing autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or APECED (125), the Forkhead-Transcription-Factor-Like 2 (FOXL2) gene causing blepharophimosis-ptosis-epicanthus inversus syndrome (126), and the Galactose-1-Phosphate Uridylytransferase (GALT) gene causing galactosemia (127) located on chromosomes 21, 3, and 9, respectively. Myotonic dystrophy is an autosomal triple repeat disorder, like the fragile X premutation carrier state, that is similarly associated with premature loss of germ cells from the ovary. Autosomal genes for which mutations have been associated with nonsyndromic premature loss of germ cells include Inhibin A (INHA) (another member of the TGF-β family), NR5A1 (SF1), and NOBOX (128-131). Of these, mutations in SF1 have been most commonly found, first in 46,XY gonadal dysgenesis patients, and more recently in patients with primary and secondary amenorrhea with 46,XX ovarian insufficiency (130). Other autosomal candidate genes are currently under study (e.g., DAZL). It would appear that all of these mutations cause loss of germ cells.

Most previous studies have focused on single gene mutations and POI. However, it is likely that some etiologies of POI are multifactorial in nature with synergism between different genes and other epigenetic factors, particularly in complex diseases such as POI. After a recent GWAS study found associations between ADAMTS19 gene mutations and POI, further interest in the role of ADAMTS genes in ovarian development and function spurred additional genetic studies. (132) ADAMTS is expressed in the embryonic phase of gonadal development being important for angiogenesis and organ morphogenesis. SNP analysis found significant epistasis between SNPS in IGF2R and specific diplotypes for ADAMTS19 in women with POI. The authors hypothesized that since IGF2R is important in steroidogenesis and ADAMTS genes are regulated by progesterone, women with SNPs and diplotypes for these two genes are at higher risk of POI.

Our molecular understanding of hypergonadotropic hypogonadal patients has revealed a number of patients with seemingly normal ovarian development for whom germ cell depletion is not the cause of the elevation of gonadotropins. Rather, in these patients, the inability for steroid production is usually the cause of the hypergonadotropic state; and, hence the classification of ovarian insufficiency. The first such classic syndrome, Savage syndrome, was originally described as gonadotropin resistance. Initially, a number of families were identified in Finland in which 46,XX individuals with gonadotropin resistance were found to be homozygous for a single mutation of the FSH receptor gene (133-135). Reports of additional mutations have since accumulated throughout other parts of the world (136,137). Subsequently, other 46,XX hypergonadotropic patients have been identified with mutations in the LH receptor (138-142), the FSH b (143,144), and the LH b genes. Overall the result of these disorders is a lack of estrogen production and variable hypergonadotropic states. 46,XY individuals with homozygous or compound heterozygous mutations of the LH receptor gene do not masculinize in-utero and present during adolescence with a female phenotype, delayed puberty, and hypergonadotropic hypogonadism. Their gonads, however, are testes not ovaries.

The second classic hypergonadotropic state that has long been described in association with otherwise normal gonads is 17 a-hydroxylase deficiency. Both 46,XX and 46,XY individuals present with delayed puberty and a female phenotype, and ovaries and testes, respectively. Mutations have been also found in this gene (145,146). Similarly, mutations of the aromatase gene in 46,XX individuals have been identified and associated with delayed puberty and hypergonadotropism in these individuals (14,147-149). In contradistinction to the other hypoestrogenic syndromes, aromatase deficiency, however, is associated with elevations of androgens in-utero and at puberty and the predictable but variable degrees of masculinization in these otherwise phenotypic females. Finally, the fascinating report of a 46,XY patient identified with a mutation in the CBX2 gene suggests a new syndrome for which hypergonadotropism is associated with seemingly a normal ovarian architecture (150). When reported, this child was under 5 years of age. A more recent cohort series of 47 patients with disorders of sexual development did not find any pathogenic mutations in CBX2 mutations within their subjects. It is likely that mutations in CBX2 are a rare cause of gonadal disorders of sexual development.(151)

46,XY Gonadal Dysgenesis.

Our understanding of Swyer syndrome (46,XY gonadal dysgenesis/sex reversal) together with 46,XX sex reversal helped to identify the SRY gene on the Y chromosome short arm (152). Common thought has held that SRY expression is the essential signal in the process of testicular morphogenesis. Hence, SRY has been seen as the master switch for this process. However, only 15% of women with 46,XY gonadal dysgenesis have been found to harbor mutations in this gene (153,154). The fact that the remaining 46,XY gonadal dysgenesis patients have intact Y chromosomes and that most 46,XX true hermaphrodites studied have not been found to harbor SRY sequences provides evidence that other genes are present and necessary for testicular development either upstream or downstream in expression to SRY. Such conjecture has been replaced with an ever growing list of now known genes operative in this pathway of testicular morphogenesis. Mutations of the WT1 (155,156), SOX9 (155,157-160), DSS (161), SF-1 (114,162), DAX-1 (160), Desert Hedgehog (DHH) (113,163), TSPYL1 (164), and CBX2 (150) genes have all been associated with specific syndromes and 46,XY sex reversal. Of these, the most frequently reported and best characterized involves the SOX-9 gene and the accompanying syndrome of Campomelic dwarfism.

Contemporary Issues for Management

Patients identified with ovarian insufficiency will need evaluation for associated medical disorders. For Turner syndrome, the most commonly identified acquired medical condition is thyroiditis. For them, the most dangerous abnormalities involve cardiovascular malformations. While previously it has been well known that coarctation of the aorta occurs more frequently for these patients as does bicuspid aortic valves, it is now evident that these patients are also at increased risk of developing dilation of the ascending aorta (and less commonly at other vascular sites) with subsequent dissection and, if undiagnosed and untreated, rupture. Like patients with Marfan syndrome, they appear to have cystic medial necrosis as the predisposing vascular histopathology. Similar to Marfan syndrome, the increased cardiovascular demands of pregnancy also appear to increase significantly this risk. The NIH consensus panel has suggested that all Turner patients have a baseline echocardiogram and, if normal, then a cardiac MRI (98). Additional evidence suggests that the MRI measurements of the aorta should be normalized for body surface area (99). Subsequent studies should be repeated every 3-5 years and perhaps during each trimester of pregnancy if patients are willing to take a risk estimated to be at least 2% for maternal mortality and, for those who survive a potentially increased risk after exposure to pregnancy.

All Turner patients should be counseled about their increased risk of dilation, dissection and rupture of the ascending aorta that is increased with pregnancy. Since most previous deaths occurred after misdiagnosis, Turner patients should be counseled to make health care providers aware of this possible diagnosis when being evaluated for disproportionate symptoms of indigestion and upper abdominal or chest pain. During dissection, the patient may have abnormal phonation and experience unusual coldness and sensations in their legs. It is possible that most deaths could have been avoided with timely diagnosis and surgical repair. Turner syndrome patients need evaluation for horseshoe kidney and for other less frequently diagnosed autoimmune disorders such as diabetes, hypertension, dyslipidemia, and hearing impairment (98).

Treatment of patients with Turner syndrome includes not only hormone replacement for pubertal progression and health maintenance at least through age 50 years, but an even earlier consideration for growth hormone treatment. While there were some initial conflicting reports, general consensus is that the use of growth hormone for enhancing adult stature is a worthwhile endeavor (165-178). The initiation of estrogen therapy at an age concordant with normal endogenous ovarian production (i.e., at least by ages 9 to 11 years) has always been considered important for normal psychosexual development of the adolescent. However, it is also believed that such early estrogen replacement might also result in an earlier closure of epiphyses and a potential limitation of final adult stature. The use of growth hormone therapy initiated during the childhood years may allow a more normal childhood stature (concordant with mid parental height) and the earlier initiation of estrogen therapy obviating these concerns (168,179,180). Synergistic benefits of low dose estradiol and GH treatment for these patients when begun as early as 5 years of age can add 2.1 cm to adult height, beyond the 5cm gain expected from GH therapy when combined with estradiol at 12 years of age. However, inappropriate feminization at a young age and unknown long term consequences of early estradiol supplementation limit the widespread use of adding estradiol to GH therapy in these young patients.(181) Other techniques to increase adult height include the delay of pubertal induction or the addition of oxandrolone until 15 years of age. Studies suggest that this technique can add up to 4cm of adult height but also raise concerns about effects of delayed puberty on bone health and the psychologic impact of delayed secondary sex characteristics.(174,182-184) Oxandrolone, an anabolic steroid, has significant side effects such as virilization and liver dysfunction, limiting its use.(185)

Most women with TS build their families utilizing oocyte donation due to premature oocyte depletion and ovarian insufficiency; an increasing number of them with gestational carriers due to the risk of death from aortic dissection (2%) during pregnancy. (186) A Nordic cohort of 106 women with TS who had a live birth after donor oocyte IVF reported 20% risk of preeclampsia, and potential life threatening complications in 3.3%, however no deaths occurred. The one woman with an aortic dissection had normal pre-pregnancy imaging. 9% of this cohort had a known cardiac defect before pregnancy. (107)

Due to the potential delayed depletion of oocytes in some TS women, there may exist a potential for fertility preservation in those women with regular menses. Case reports document the feasibility of oocyte cryopreservation in post pubertal girls (ages 13-15) with Turners syndrome who already had evidence of diminished ovarian reserve. A range of 4-13 oocytes have been cryopreserved. (187-189)

Given all of these considerations for natural reproduction in these women with TS, counseling is critical to provide them the most accurate information regarding risks and benefits. Turner syndrome remains a relative contraindication to pregnancy, and if risk factors are present it becomes an absolute contraindication. Until better data are available or prophylactic treatment of the aorta is developed that provides protection for pregnancy, counseling should be provided that includes alternatives such as use of a gestational carrier or adoption.

Patients with 46,XX gonadal dysgenesis should be evaluated for premutations of the fragile X (FMRI) gene. This finding should prompt counseling for themselves and other family members and prohibit use of their similarly affected sisters as oocytes donors. In addition, 46,XX ovarian insufficiency patients should be screened regularly for the development of Hashimoto thyroiditis and at least at baseline for adrenal steroid cell or 21-hydroxylase antibodies. Continued surveillance should be considered for the presence of hypoparathyroidism, adrenal insufficiency, and other autoimmune disorders such as pernicious anemia. All gonadal dysgenesis patients with a Y cell line need extirpation of their gonads including Turner patients with 45,X/46,XY (or those with a Y chromosome fragment) gonadal dysgenesis and the 46,XY gonadal dysgenesis patients. One should remember that rare Turner patients with seeming a single 45,X cell line might have undetected mosaicism for a Y cell line. Screening 45,X single cell line patients and those individuals with an unidentified chromosomal fragment with Y-DNA centromeric probes may be prudent to uncover those additional individuals at-risk for gonadal malignancies.

All patients with premature gonadal failure need estrogen therapy for initiation and completion of pubertal progression and subsequently for the maintenance of a multitude of health processes. While the continued accrual and remodeling of bone is of utmost importance, it remains likely that numerous other physiologic processes are dependent on normal estrogen status as well, at least through 50 years of age. The findings and concerns for long term hormone replacement of the Women’s Health Initiative do not apply to these or any other patient prior to the age of 50 years and should not be used to prematurely stop their hormone replacement.

Counseling is of utmost importance for these individuals and should cover expectations for all aspects of these young women’s lives including alternatives for reproduction. While the use of donor oocytes and IVF has proven safe for 46,XX and 46,XY gonadal dysgenesis patients, an estimated maternal death rate of at least 2% exists for Turner syndrome patients and pregnancy may increase the risk for rupture in future years. While it is often easier to include pregnancy by donor oocyte as an alternative during counseling, until more information is available such discussions should be framed with the above concerns. One should also turn to patient guidelines of national organizations such as the American Society for Reproductive Medicine (ASRM) and the American College of Obstetricians and Gynecologists (ACOG) as they are developed about these issues. The use of “buddy programs” in which these patients are paired with others who have previously confronted the same issues during adolescence and support groups (e.g., Turner Syndrome Society) is an excellent complement to this counseling.

Hypogonadotropic Hypogonadism

A number of young women will present with delay of the onset of pubertal development who have no evidence of ongoing estrogen production, because something has interrupted either GnRH or gonadotropin secretion from the hypothalamus/pituitary. Patients with constitutional delay of puberty represent the most common of these disorders. Other disorders are clearly congenital or acquired.

Constitutional delay

Constitutional delay of puberty refers to a common condition for which patients will go through puberty but at a time that is more than 2.5 standard deviations delayed from the mean (Tables III and IV) (83-85). A number of these patients often have a family history of delayed puberty (85). Their physiologic age (i.e., bone age) lags behind that of their peers and is manifested by a delay in the adolescent growth spurt and temporary short stature. Most of these patients present between 13 and 16 years of age and at that time have very early signs of thelarche. Their gonadotropin levels are in the low to normal range and their workup is otherwise unrevealing.

Until recently, no specific mutations had been identified as causing constitutional delayed puberty, despite the observation that 50-80% of those with this disorder have a positive family history. A recent proband study evaluated families with delayed puberty for some of the common mutations found in idiopathic hypogonadotropic hypogonadism ( IHH). They found that subjects with constitutional delayed puberty more commonly shared the same mutation with affected family members compared to non-affected family members (53% vs 12%, p = 0.03). They even found that subjects with delayed puberty without a similar family history were more likely than controls to carry mutations commonly seen in IHH (14.3% vs 5.6%, p =0.01)(190).

In males, 60% of pubertal delay is constitutional. In females, however, no more than 30% have this benign reproductive condition. While constitutional delay represents a leading cause of female pubertal delay, prior emphasis on this statistic has led to the false diagnosis for many young women and the misguided reassurance that they were simply “late bloomers.” As many as two-thirds of females presenting with delayed puberty will have an irreversible etiology for reproductive failure, not constitutional delay (83). For this reason, any patient presenting with delayed puberty and given the label of constitutional delay should be scrutinized very carefully for other etiologies, especially if they are beyond age 16 years and have yet to initiate pubertal development.

It can be challenging to differentiate constitutional delay from IHH. Given the finding of similar mutations observed in some of both groups of patients, these disorders may, in fact, fall in the same spectrum, one being reversible and the other not. Numerous tests have been proposed to help distinguish the two; however, none have been particularly helpful. When previously performed, an intravenous GnRH challenge test usually confirmed early awakening of the hypothalamic-pituitary-ovarian circuit by demonstrating a pubertal gonadotropin response, i.e., a greater release of LH than FSH. Such a response is seen only after endogenous GnRH secretion occurs and puberty is at or beyond its very early stages. At the same time, this early gonadotropin release produces the multifollicular ovarian appearance of early puberty; the ultrasound appearance of which is likely as reassuring that puberty is marching onward as is the LH response of a GnRH challenge. The most helpful distinction between IHH and delayed puberty is the failure to enter puberty by the age of 18 years. However, many patients and their parents may not readily adopt the wait and see tactic and instead may prefer additional periodic assessments. One option is to follow with pelvic ultrasound studies looking for the appearance of the multifollicular ovary associated with the early stages of pubertal progression. It would be ideal that no adolescent would reach mid teenage years without spontaneous or exogenously-induced pubertal development!

Acquired Abnormalities

A number of acquired medical conditions may interfere with either the production of GnRH and/or gonadotropin secretion producing a hypogonadotropic hypogonadal state (Tables III and IV) (83,85). The Children’s Hospital series refers to many of these as functional disorders (85). Endocrine disorders such as hypothyroidism, congenital adrenal hyperplasia, Cushing syndrome, and hyperprolactinemia that begin before or during the early pubertal process may interfere with gonadotropin secretion. While only some cases of growth hormone deficiency are acquired, this disorder is included here with the other endocrinopathies. Patients with unusually short stature, pubertal delay, and low gonadotropin levels should be considered as having one of the endocrinopathies that also affects growth (i.e., hypothyroidism and growth hormone deficiency). Treatment of these disorders will allow the resumption of puberty. Systemic illnesses including malabsorption states, eating disorders, active autoimmune diseases, and the rare hypoxemic states related to congenital heart malformations or severe anemias (i.e., sickle cell) are also occasionally etiologic for hypogonadotropism and pubertal delays. Most of these conditions are similarly reversible. Finally, pituitary tumors are consistently reported in rare patients of all descriptive delayed puberty series (83). The craniopharyngioma occurs usually between the ages of 6 and14 years prior to the usual time onset of puberty. It is an aggressive tumor that causes early destruction of the pituitary and suprasellar regions and usually delays any pubertal development. On the other hand, it can also be an indolent tumor not becoming apparent until the late teenage years or even the mid 20’s. The typical calcification of these tumors makes them easily diagnosed radiologically. Unlike the craniopharyngioma, the prolactinoma usually does not develop until after puberty is initiated. Estrogen is known to increase messenger RNA for prolactin and its increase at puberty is seemingly associated with the development of prolactinomas in at-risk individuals. For these patients, the prolactinoma usually arrests a pubertal process that has begun on time. These tumors are extremely slow growing and rarely interfere with other pituitary functions, if at all. If a dopamine agonist is given to lower the prolactin levels, puberty or menstrual function will usually proceed normally. The prolactinoma now outnumbers the craniopharyngioma as a cause of hypogonadotropic hypogonadism (83).

Congenital Abnormalities

A number of disorders classically felt to be irreversible are found in patients with hypogonadotropic hypogonadism. Some of these patients present with fractional or complete pituitary insufficiency. The majority of patients have been historically categorized with idiopathic hypogonadotropic hypogonadism (IHH) and, despite the fact that specific causes have now been identified for as many as 30% of them, the label of IHH has persisted. Such patients have absence of spontaneous pubertal development that persists beyond age 18 years; hypogonadotropism is usually the isolated pituitary deficiency for them. Specifically they have functional GnRH deficiency. Numerous studies involving frequent blood sampling have demonstrated 4 different aberrant patterns of gonadotropin secretion. The majority of patients with IHH demonstrated apulsatile secretion and the remainder were divided between sleep entrained pulsitility, decreased pulse frequency, and decreased pulse amplitude (191).

Both syndromic and nonsyndromic etiologies exist. Kallmann syndrome (KS) refers to IHH with anosmia or hyposmia. The association of IHH with anosmia is not surprising given that the GnRH secretory neurons originate within the olfactory placode and then migrate to the hypothalamus extending their axons to the median eminence. Normosmic IHH (nIHH) refers to those IHH patients with a normal sense of smell. A number of genes have been identified that regulate development and migration of GnRH neurons, the production, processing and secretion of GnRH, and its expression at the receptor. (192) Mutations have been identified within these genes which result in both KS and IHH and will be discussed further in this chapter. X-linked KS and some of the patients with mutations in these other genes may have unilateral renal agenesis (KAL1 mutations in males), midline facial defects, or neurologic and skeletal abnormalities (193,194).

It has always been intriguing that variable phenotypes have existed within families harboring the same IHH mutation (193-195). Perhaps more intriguing have been the reports that 10% of males with IHH, some with mutations within genes regulating GnRH neuronal development or secretion, have reversal of their disorder and spontaneous continued reproductive function after discontinuation of treatment that may have been given for months or years (196). Recent studies of adult onset hypogonadotropic hypogonadism in males with prior reproductive function have also reported finding the same mutations shared by those men who never initiated puberty. A series of 32 male patients with hypogonadotropic hypogonadism were assessed after treatment withdrawal and 6% had recovery of gonadal function.(197) Similarly, reversible hypogonadotropic hypogonadism has been reported after years of treatment in women, one who also had anosmia (personal patient, reported only in abstract form, Goldstein, Fertil Steril 2011,96: S116, ), and an adult onset form has been identified in women with hypothalamic amenorrhea sharing similar mutations.(198) Taken together, with the prior information about similar mutations in some patients with constitutional delay of puberty, what was previously labeled as IHH appears often to be a part of a spectrum disorder with overlap between constitutional delay of puberty (spontaneous early resolution), irreversible forms in both males and females, late onset forms in individuals who first established reproductive function, and late reversible forms in patients with prior longstanding hypogonadotropic hypogonadism. All of these forms of hypogonadotropic hypogonadism have been shown to have some patients with mutations in the same genes.

A number of other genetic defects have been found to cause hypogonadotropic hypogonadism such as leptin deficiency and adrenal hypoplasia congenital (193,199-206). Besides IHH, forms of hypopituitarism also exist and result in delayed puberty with hypogonadotropism. Included are septo-optic dysplasia (SOD) (207,208), combined pituitary hormone deficiency (CPHD) (209-212), CHARGE syndrome (213,214), Prader-Willi Syndrome, and Laurence-Moon-Bidel-Bardet Syndromes. Finally, other forms of hypopituitarism exist, some of which are associated with anatomic abnormalities such as Rathke’s pouch cysts, anterior encephalocele, and hydrocephalus (83).

Molecular Findings

As in the patients with hypergonadotropic hypogonadism, molecular research has provided new insight into the clinical findings of a number of patients with hypogonadotropism. In particular, these studies have helped to better understand the variation of clinical presentation and gonadotropin levels, and the different responses to exogenous GnRH reported in these patients. For men with Kallmann syndrome, the first mutations found were those involving a cell surface adhesive gene, the KAL1 gene (215-217). The initial identification of these mutations began our understanding of the anosmia and hypogonadotropic state for KS patients; such mutations prevent normal development of the neurologic tract responsible for transport of GnRH to the median eminence and the olfactory bulb (193,218-221). Subsequently, a number of these men were also found to have unilateral renal agenesis. While similar mutations have not yet been identified in anosmic females, it is likely that a few will ultimately be uncovered. The second molecular finding involved nIHH patients and was the identification of mutations in the GnRH receptor gene (222-225). Since then, a host of mutations have been identified in hypogonadotropic patients; genes involved generally have their adverse effects in the hypothalamus, pituitary, or both.

Hypothalamic defects that are etiologic for KS and/ or nIHH involve mutations in genes responsible for GnRH production (GNRH1 gene) (226), GnRH processing (PCSK1 gene) (227-229), GnRH neuronal development that prevents subsequent normal transport through the neuronal pathways to the median eminence [FGFR1 (215,230-235), FGF8 (236), PROK2, PROKR2 (237) and CHD7 (238) genes in addition to the KAL1 gene], and GnRH secretion (GPR54 or KISS1 and receptor genes) (33,34,190,239,240) into the portal circulation.

Those genes for which mutations have been identified as a cause of IHH primarily at the level of the pituitary include the GNRHR, HESX1 (207,208), PROP1 (209,210,241), SOX2 (242), SOX3 (243), LHX3 (211,212), LHX4 (244,245), LHβ (246), and FSHβ (144) genes. Except for GNRHR or gonadotropin β gene mutations, the other mutations produce a host of phenotypic findings that often include other pituitary or endocrine deficiencies. Mutations within the leptin (201,202), leptin receptor (204,206), and NROB1 (DAX1) (247,248) genes appear to cause IHH within both the hypothalamus and pituitary. The former mutations are associated with extreme obesity (201,202). Finally, additional mutations yet to be fully understood have been found in IHH patients in the TAC3, TACR3, and nasal embryonic LHRH factor (NELF) genes (249). Numerically, the most commonly found mutations among IHH patients are those within KAL1 (men only), the FGFR1, CHD7 (CHARGE syndrome), and GNRHR genes. Interestingly, the least common and last to be identified are the mutations in the GNRH gene.

The identification of all of these mutations gives us tremendous insight into the requirements and signals for normal pubertal development It appears that the pubertal process is well orchestrated between a number of different genes and a mutation in any one of them may result in the absence of pubertal development. Given the findings of KISS1 and KISS1R mutations in a few patients with central precocious puberty, if there is a single signal for the pubertal process among all of the genes identified it is likely kisspeptin. The other genes identified in these patients with hypogonadotropic hypogonadism appear to provide the framework within which the reproductive system works. We now know that a number of genes are involved in laying down the normal neuronal transport pathway for GnRH. Some are sufficiently tightly involved with the optic bulb development (KAL1) that all patients with mutations have anosmia. Mutations in others (FGFR1) may result in either anosmic or normosmic IHH. It also appears that if a mutation exists in one of the genes that prevents normal neuronal development (e.g., FGFR1), rarely sufficient development may ultimately occur in the absence of this seemingly critical protein either with time or induced from hormone therapy such that reversal of this disorder may occur in a few patients (196). There seems to be overlap between these genes as well, given that patients may be compound heterozygotes with two mutations and each in a different gene (249). In addition, several patients have presented with a KS-like phenotype and found to have mutations in CHD7 gene, usually etiologic for the CHARGE syndrome (238).

Contemporary Issues for Management

As has been elaborated, numerous different disorders exist for patients presenting with hypogonadotropic hypogonadism. Many of these are rare and best managed by specialists who treat the specific disorder, each disorder having very specific individual clinical concerns. It should be determined early whether treatment of the disorder will allow subsequent pubertal progression or whether a form of hypogonadotropism exists for which puberty will not progress without sex steroid replacement. Early hormone therapy is critical for the management of such patients. Similarly important is the individual counseling about expectations for pubertal development, associated problems, reproductive options, and chance of recurrence or reversal. No doubt, this may require a multidisciplinary team approach. An interesting finding of the Children’s Hospital study was that it provided evidence that there may be an association between hypogonadotropic hypogonadal causes of delayed puberty and attention deficit disorder with or without hyperactivity (85). Finally, as more and more gene mutations are identified in IHH patients, an understanding of minor phenotypic findings associated with them may make earlier diagnosis possible. When seen, for example, in an extremely obese adolescent, leptin or leptin receptor mutations should be considered.

Eugonadism

The MCG series presented a third group of females with pubertal abnormalities and evidence of ongoing estrogen production. These patients primarily present with delayed menarche.

Anatomic Abnormalities

Congenital absence of the uterus and vagina (CAUV), also known as müllerian aplasia or Meyer-Rokitansky-Kuster-Hauser-syndrome (MRKH), is the second most common cause of pubertal aberrancy in the MCG series (84). In particular, these patients present with delayed menarche. They have fusion failure of the two müllerian anlagen during embryogenesis. The normal fusion process is usually followed by canalization of the vagina. In its absence, small uterine remnants and their attached normal fallopian tubes remain; the vaginal plate and uterine remnant(s) are uncannalized. Rare patients will have a variable degree of uterine fusion and/or variable foci of functional endometrium (250). These patients progress through puberty at the normal time. They present with normal pubertal development and delayed menarche and on examination are found to have isolated absence of the vagina. They have normal ovarian function. Nearly 30% of these patients have concomitant renal abnormalities, including unilateral renal agenesis, horseshoe kidneys and urethral duplication. From 12 to 50% of these patients will have associated skeletal abnormalities, scoliosis being the most common and limb defects such as lobster claw hand deformity and phocomelia rarely present (83). Other abnormalities may also occur.

Another group of patients who may present with an anatomic cause of delayed menarche are those with an imperforate hymen or rarely a transverse vaginal septum (TVS). Given the average age of menarche, most girls with an imperforate hymen will present several years before the age of 15 years and thus may not be “labeled” as presenting with primary amenorrhea. While a complete TVS causes a presentation similar to imperforate hymen, the majority of patients with a TVS will have perforations in their septum and will not present with absence of menses.

Patients with an imperforate hymen or complete TVS initiate puberty at the normal time and present with cyclic pain, on average, within 1 to 2 years after menarche. Being obstructed, they develop an hematocolpos with or without an hematometra. On examination they are found to have an obstructing membrane, the thin imperforate hymen often bulging on valsalva maneuver or a thicker TVS. The latter is usually located at the junction of the upper one-third of the vagina but can be at lower levels as well and because of its thickness usually does not bulge on valsalva. Once these obstructing membranes are surgically excised normal menstrual function usually follows. In contrast to patients with outlet obstruction, those with vaginal agenesis will usually have normal hymeneal tissue and either an absent vagina or a small pouch created by attempts at coitus. For them, there is never a midline mass on rectal exam.

Molecular Findings

Because patients with CAUV were never previously able to have children, the inheritance pattern for most of them has been generally unknown and clues for potential candidate genes have remained elusive. The majority of these patients are sporadic occurrences within their family. Rare sibships with several non-twin sisters affected have been reported and twins both concordant and discordant for CAUV also exist (83). A report of the outcome of pregnancy for these patients who were able to have their own biological children through IVF utilizing a gestational host suggests that this condition is not commonly autosomal dominant; none of the female babies were found to be similarly affected (251).

A number of genes have been proposed as candidate for harboring germ-line mutations etiologic for the syndrome of CAUV. The anti-Müllerian hormone (AMH), anti-Müllerian hormone receptor (AMHR), and other genes involved in the pathway of AMH directed müllerian regression (e.g., the β-catenin gene) have been considered likely candidates. Since a number of somatic systems are involved in this syndrome, studies have centered around developmental genes and in particular, the HOX family of genes. In addition, HOXA10 is expressed in the developing paramesonephric ducts. Mutations in HOXA13 have been associated with the hand-foot-genital syndrome and in HOXD13 have caused synpolydactyly in humans. Furthermore, the PBX1 gene protein is thought to be a HOX cofactor during müllerian and renal development. Other developmental gene candidates considered have included the PAX2, Wilms tumor transcription factor (WT1), and WNT4 genes as well as genes controlling the synthesis of retinoic acid receptors, the RAR-gamma and RXR-alpha genes. The latter 3 of these genes, when mutated in mice, have produced müllerian abnormalities. Finally, given that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations cause congenital absence of the vas in men and that the early wolffian anlagen seemingly direct müllerian development in females, this gene too has entered the list of suspects.

Our laboratory has performed mutation analyses for a number of these candidate genes in müllerian aplasia patients including CFTR (252), WNT7, AMH (253), AMHR (253), HOXA10 (254,255), HOXA13 (256), galactose-1-phosphate uridyl transferase (GALT), PAX2 (257), WT1 (258), and WNT4 (259). Studies by others have not found mutations in HOXA7, HOXA13, PBX1 (260), β-catenin (261), RXR-α, and RXR-γ genes (262). To date worldwide, excepting WNT4, none of these analyses have revealed a convincing association.

Several patients with congenital absence of the uterus and vagina have now been identified with mutations in the WNT4 gene (263-265). These patients all seem to have a variation of the classic presentation of congenital absence of the uterus known as Mayer-Rokitansky-Kuster-Hauser syndrome. In addition to müllerian aplasia, these patients have signs or biochemical evidence of androgen excess and either modified location of their ovaries (in two patients) or seemingly hypoplastic ovaries (in the third patient). Their phenotype is very similar to that of the WNT4 knockout female mouse: absence of the müllerian system associated with aberrant androgen overproduction and premature loss of follicles (266). Studies of these 3 patients have given further insight into the role of WNT4 in human reproductive development and steroidogenesis. Given the infrequency of these mutations in patients with congenital absence of the uterus and vagina, however, some have proposed that it is, in fact, a specific entity (264,265,267). A study of ovarian steroidogenesis and oocyte number in patients with müllerian agenesis undergoing IVF for transfer of embryos to a gestational carrier did not find impairment in either of these parameters (268). This further supports that WNT4 mutations are rare and a specific entity.

With the development of next generation sequencing and its ability to investigate genetically heterogeneous diseases, whole exome sequencing is utilized for diseases for which causative genes have not yet been identified. A recent whole exome sequencing and copy number variation case series in women with MRKH showed high frequency in loss of function variants of the OR4M2 and PDE1 1A genes and deletions in 15q11.2, 19 q13.31, 1pq36.21, 1q44, suggesting new candidate genes in the development of MRKH. (269)

One may question why, except in a rare phenotype that seems to be a different entity (i.e., patients with WNT4 mutations), no individuals with classic Mayer-Rokitansky-Kuster-Hauser syndrome have been found to harbor a mutation in a host of very likely candidate genes? Explanations might include: (1) the presence of mutations in yet-to-be-studied candidate genes; (2) multifactorial inheritance; or, (3) the presence of nonconventional genetic mechanisms. The latter seems to be an attractive explanation. In particular, this condition has the characteristics of disorders such as McCune-Albright Syndrome that are caused by somatic cell rather than germ-line mutations; somatic cell mutations occur at some point after fertilization in the dividing somatic cells of the embryo or in stable somatic cells later in life. They are almost never present in the germ cells. As a result the patient is usually a random occurrence within a family and neither inherits nor passes this condition on to the next generation. If this occurs during development (such as seen in McCune-Albright syndrome), the mutated somatic cells will migrate to various areas of the fetus; the phenotype always being consistent, but often with some variation dependent on the final location of the affected cells. The vast majority of patients with Mayer-Rokitansky-Kuster-Hauser syndrome are the only such affected member of a family. The consistent phenotypic findings in these patients all involve the loss of structural integrity (müllerian aplasia, renal agenesis, and bone defects) and some degree of variability exists with which specific system is involved. Patients with scoliosis, lobster claw defects and congenital amputations represent the extreme variation. Somatic cell mutations would easily explain each of these occurrences. The report of identical twins, one with isolated vaginal agenesis and the other with bilateral tibial longitudinal deficiency (congenital leg amputations) (270) makes a strong case that somatic cell mutations beginning in the initial embryo migrated to the bones in one twin and to the developing müllerian system of the other, after the process of identical twinning. Unfortunately, if, in fact, somatic cell mutations are etiologic for most cases of müllerian aplasia and involve genes that cause loss of structural integrity, the cells with the culprit mutations may no longer be present for analysis. They may have been in the original cells of the now absent uterus, vagina, kidney or bone. A recent comparative study of different tissues (blood, saliva, rudimentary uterus) in 5 pairs of discordant monozygotic twins found differences in copy number variations utilizing SNP microarray technology in the affected twin compared to non-affected twin in the following genes: MMP-14, LRP- 10, ECM, and neoangiogenesis genes. There were no differences between the mutation analyses in saliva, but similar differences in the blood and uterine tissue, mesodermal derivatives, suggesting a tissue specific mosaicism.(271)

For the transverse vaginal septum and imperforate hymen patients, molecular analysis has been essentially nonexistent.

Contemporary Issues for Management

The diagnosis of CAUV is essentially clinical. The classic finding of absence of the vagina or a vaginal pouch (usually developed through prior coital attempts) associated with otherwise Tanner stage 5 breast and pubic hair development is unlikely anything else but CAUV. A search for associated physical findings of bony malformations (commonly scoliosis) and rarely inguinal hernias or scars from prior repair should be conducted. The inguinal hernias occur because the round ligaments can pull the unconnected uterine remnants and associated fallopian tubes and ovaries into the inguinal canals. The diagnosis of CAUV can be confirmed simply by a pelvic ultrasound study that demonstrates the presence of ovaries with follicular activity. The midline uterus will not be seen. Neither a karyotype nor laparoscopy is necessary for the diagnosis in the majority of CAUV patients. The prepubertal patient could be misdiagnosed with AIS. However, post-pubertal the clinical findings for CAUV and AIS are sufficiently different that diagnosis of each is usually straightforward. If in doubt, a serum total testosterone level is the least expensive method of resolving the confusion; levels within the female and male ranges will differentiate the two conditions. One must now always consider, however, the WNT4 mutation syndrome for which patients with müllerian aplasia may manifest symptoms or biochemical evidence of androgen excess and reduced ovarian reserve.

Although not currently recommended as first-line management, treatment of this condition has previously been surgical; a number of different surgical techniques have been utilized for creation of the vagina. In the United States, the McIndoe vaginoplasty has been the most commonly performed surgery for neovaginal creation. This is the classic procedure in which a skin graft is sewn around a mold and inserted into a newly dissected vaginal space. After a skin graft takes, the patient wears a vaginal mold for an extended period of time and until regular coitus to prevent scarring down of the neovagina. In other parts of the world and some areas of the US, the Vecchietti procedure is more commonly performed. In this procedure an olive shaped instrument is placed at the perineal dimple and pulled inward under tension by attached wires, sutures, or threads stretching the perineal skin in the direction of the normal vaginal axis. The tension cords were originally placed by abdominal surgery and in more recent years have been placed by laparoscopy (272-276). Another procedure, the Davydov procedure, was developed in Russia and is gaining popularity worldwide including the US (277,278). In this procedure, laparoscopic assistance is used to bring peritoneum from the pouch of Douglas into the space created for the neovagina. A purse-string suture is placed at the top and the neovagina is created. Results of both of these alternatives have been overall very encouraging (279-281).

The majority of patients, however, can avoid surgery altogether and should be encouraged to attempt creation of a neovagina first by the Ingram dilation technique (282,283). Experts have agreed that the nonsurgical approach should be the first line approach because it is successful in approximately 90% of patients, is less morbid than surgery, and is not associated with possible contracture (284,285). A vaginal dilator is held in place at the vaginal dimple with athletic underwear. The patient then sits on a bicycle seat of a stationary bicycle or a specially designed chair for regular periods of time. The size of the mold is increased over time and until a normal sized vagina is created or coitus can be initiated. With motivated patients and careful instructions and follow-up the majority of patients will succeed. When new patients are paired up with prior successful CAUV patients for support, this method rarely fails. Patient pairing is particularly helpful for the emotional support and personal advice that only women who have weathered the various challenges of this condition can provide.

The assisted reproductive technologies have provided these women a means of having their own biological children. The use of gestational carriers with IVF after oocyte retrieval and fertilization has made this possible. Given that the CAUV patient and her husband are the biological parents of these children, legal issues involving the gestational carriers are certainly better delineated and problems arising from them much less likely than were the initial uses of surrogacy.

Recent advances in uterine transplantation have led to the first live born infant and several additional pregnancies from a transplanted uterus to patients with MRKH. The Swedish team spent years of preparation and experimentation beginning with animal models understanding basics of the surgeries involved as well as immunosuppression. They developed separate teams for removing and implanting the uteri. In some parts of the world, including Sweden, the use of gestational carriers is banned. As a result, uterine transplantation is the only hope in these countries for having a biological child for these women with MRKH. Since this therapy remains highly experimental and fraught with both medical and ethical concerns regarding potential surgical complications as well as issues from immunosuppression, it should only be performed by teams as well prepared as the Swedish team, who has completed this remarkable feat.

Counseling patients with vaginal agenesis and other disorders of sexual development (DSD) requires special skills and sensitivities and is covered briefly at the end of this chapter.

The imperforate hymen and the transverse vaginal septum are surgically treated by one of a number of procedures described in most gynecologic textbooks. These procedures are usually straightforward. Occasionally the transverse vaginal septum is difficult and requires more involved surgery including an abdominal approach, a Z-plasty or skin graft. None-the-less, only an experienced surgeon should perform all of these procedures.

CHRONIC ANOVULATION

Polycystic ovarian syndrome (PCOS) and a number of other endocrine abnormalities may result in chronic anovulation and may present as delayed menarche as reported in the MCG series (83). Although most patients with PCOS present in adolescence with menstrual irregularity, occasionally a patient will present with primary amenorrhea. If patients are androgenzied and have not menstruated they should be evaluated by at least age 14 years as covered above. These patients may not have their first menses until given a progestin challenge. While most of them have classic PCOS, other endocrinopathies and hypothalamic dysfunction need to be ruled out. The contemporary management of PCOS and its associated gynecologic and metabolic disorders includes evaluation for diabetes and hyperlipidemias and consideration for treatment of it as an insulin resistant state in addition to the classic management considerations of ovarian suppression, endometrial protection, as well as androgen targeted treatments. This topic is covered in greater detail elsewhere in this text.

DISORDERS OF SEXUAL DEVELOPMENT

Patients with androgen insensitivity present at puberty with normal onset of breast development, absent pubic hair, and delayed menarche. These 46,XY women have been found to harbor mutations in their androgen receptor genes that render their androgen receptors nonfunctional. Despite normal testes development and normal male testosterone production, they are unable to convert the testosterone signal into the end organ events of masculinization of the external genitalia in-utero or at puberty. They present with a normal female phenotype and a small blind vaginal pouch. At puberty, their androgens are converted to estrogens with normal breast development. They are usually taller than predicted by mid-parental height for females because of the presence of the Y chromosome and its associated statural genes. The presence of the Y chromosome places them at risk for developing malignancies of their gonads and dictates removal. Unlike gonadal dysgenesis patients, the risk does not increase until after puberty; additionally, these tumors are usually seminomas rather than the gonadoblastomas or germ cell tumors. Unless the testes are located within the inguinal canals, they are usually left in place until after breast development is complete.

Molecular Findings

Androgen insensitivity syndrome has been extensively studied by molecular analysis (286,287). A number of intriguing and frustrating findings have been made. First, mutations have been found in virtually every portion of the androgen receptor (AR) gene (288). Mutations in the hormone binding region of the AR gene have explained those classic patients previously determined to have nonfunctional androgen receptors. Mutations in the DNA binding domain helped explain why other AIS patients with the same classic phenotype had normally binding androgen receptors. Second, many families studied have mutations unique to their specific family (286). Until gene sequencing is routine, this precludes studying patients with a suspicious AIS phenotype for a specific AR mutation. Third, identification of mutations in this gene has widened the spectrum of incomplete AIS phenotypes to include phenotypic females with genital ambiguity, phenotypic males separately with undermasculinization (289), gynecomastia, breast cancer (290), prostatic cancer, or azoospermia/severe oligospermia (291). Fourth, individuals with the same mutations have exhibited varying phenotypes (288,292,293). Finally, clinical correlations have been made between specific mutations and the ability to masculinize further with exogenous androgens for those individuals with a male sex of rearing and not presenting as delayed female puberty (294,295).

Contemporary Issues for Management

For the classic patient with AIS who presents with delayed menarche, absent pubic hair, and a vaginal pouch, an expedient evaluation and diagnosis is necessary. Unlike the CAUV patients, once the diagnosis of AIS is suspected, chromosomal analysis is necessary to document a 46,XY karyotype. It is necessary to remove the gonads in patients with AIS (296). This can be done after puberty to allow spontaneous breast development. Support for this includes the fact that the earliest reported malignancy in patients with AIS is 14 years of age.

No doubt, one of the most critical issues related to this syndrome is counseling. No longer is it possible or advisable to hide the presence of the 46,XY finding from these patients. However, a multidisciplinary and well thought out approach and close follow-up is needed for such counseling. The psychosexual transition during adolescence is difficult and patients with intersex disorders/disorders of sexual development will face an even more difficult transition. Patients and their family require support and should be actively involved in the decision processes. Links to a variety of support groups for specific disorders can be found on the Disorders of Sexual Development website (296,297).

Many of these patients have a vaginal pouch, the embryonic remnant of the prostatic utricle. For them, coital attempts will enlarge the vagina and surgery is not needed. For others a similar, although somewhat different, approach can be utilized as was described for the patients with CAUV. Furthermore, once gonadectomy is performed, estrogen replacement therapy is essential for all of the obvious reasons.

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Giannesi A, Marchiole P, Benchaib M, Chevret-Measson M, Mathevet P, Dargent D. Sexuality after laparoscopic Davydov in patients affected by congenital complete vaginal agenesis associated with uterine agenesis or hypoplasia. Hum Reprod. 2005;20(10):2954-2957.
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Anatomy and Physiology

TREATMENT

SECONDARY DISORDERS OF LIPID AND LIPOPROTEIN METABOLISM AND SPECIAL POPULATIONS

GENETIC DISORDERS OF LIPID AND LIPOPROTEIN METABOLISM

BACKGROUND

Dyslipidemia in Chronic Kidney Disease

Recieved 2/23/15

Abstract

Chronic kidney disease (CKD) is associated with a dyslipidemia comprising high triglycerides, low HDL-cholesterol and altered lipoprotein composition. Cardiovascular diseases are the leading cause of mortality in CKD, especially in end stage renal disease patients. Thus, therapies to reduce cardiovascular risk are urgently needed in CKD. Robust clinical trial evidence has found that use of statins in pre-end stage CKD patients, as well as in renal transplant recipients, can decrease cardiovascular events; however, providers need to be aware of dose restrictions for statin therapy in CKD subjects. Furthermore, statin therapy does not reduce cardiovascular events in dialysis patients, nor does statin therapy confer any protection against progression of renal disease. Niacin and fibrates are effective in lipid lowering in CKD and appear to have some cardiovascular benefit but further study is needed to clearly define their role. This article reviews the epidemiology of CKD, association of CKD with cardiovascular events, and the effects of CKD on lipid levels and metabolism. The article discusses clinical trial evidence for and against statin and non-statin lipid lowering therapy in CKD patients.

CKD epidemiology

Chronic kidney disease (CKD) is defined as renal impairment greater than 3 months duration that results in an estimated glomerular filtration rate (eGFR) < 60ml/min/1.73m². CKD is classified into 5 stages based on the eGFR (Table 1). CKD is a world-wide health problem with rising incidence and prevalence. CKD, especially in the early stages is often asymptomatic; thus, the actual prevalence may be even higher than estimated. End stage renal disease (ESRD) is defined as needing dialysis or transplant, and the prevalence and incidence of ESRD have doubled over the past 10 years(1). The annual mortality rate of dialysis patients is greater than 20%. The burden of co-morbidities and the cost of caring for CKD patients is high, and thus a major focus is increased screening and early detection of CKD when interventions to delay or prevent progression to ESRD may be effective. There are multiple causes of CKD with the most common causes in westernized nations being hypertension and diabetes; however, a wide range of etiologies including infectious, auto-immune, genetic, obstructive, and ischemic injury are all prevalent. There are ethnic differences in susceptibility with increased prevalence in Mexican-Americans and non-Hispanic blacks compared to Caucasians(2).

While the burden of CKD itself is significant, the leading causes of morbidity and mortality in CKD are cardiovascular diseases (CVD), primarily atherosclerotic coronary artery disease. Risk factors for CVD in CKD include the traditional risk factors – hypertension, sex, age, smoking, and family history and CKD patients appear to benefit similar to non-CKD patients from therapies targeting these risk factors. Regardless of the cause of CKD, patients with CKD are at increased risk for CVD, which has led to the National Kidney Foundation classifying all patients with CKD as “highest risk” for CVD regardless of their levels of traditional CVD risk factors. The focus of this chapter is on the dyslipidemia of CKD and the risk of CVD in CKD.

Nephrotic syndrome:
Nephrotic syndrome differs from other types of CKD in its presentation and risks. Nephrotic syndrome is comprised of significant proteinuria (typically > 3g/24h), hypoalbuminemia, peripheral (+/- central) edema, and significant hyperlipidemia and lipiduria may also be seen. It is frequently seen in children, and the etiology includes minimal change disease (up to 85%), focal segmental glomerulosclerosis (up to 15%) and secondary causes (rare) including systemic lupus erythrematosis, Henoch Schonlein Purpura, or membrano-proliferative glomerulopathy. In adults, the etiology is more likely to involve a systemic disease such as diabetes, amyloidosis, or lupus. Nephrotic syndrome may be transient or persistent. Most (approximately 80% of children) cases of nephrotic syndrome are successfully treated with glucocorticoids with resolution of all features including hyperlipidemia; however, steroid-resistant nephrotic syndrome patients often have persistent dyslipidemia, which may place them at increased risk for CVD. For example a small study found increased CVD markers including pulse wave velocity, carotid artery intima-media thickness and left ventricular mass in patients with steroid-resistant nephrotic syndrome compared to controls(3), implying increased risk for CVD events. Treatment of nephrotic syndrome dyslipidemia includes therapies specifically targeting the renal disease (primarily glucocorticoids, but also renin-angiotensin system antagonists which can help decrease proteinuria) and lipid lowering agents.

Table 1: Stages of CKD

CKD stage	GFR (ml/min/1.73 m²)
CKD 1	≥ 90 (with renal damage or injury)
CKD 2 (mild)	60-89
CKD 3 (moderate)	30-59
CKD 4 (severe)	15-29
CKD 5 (end stage)	<15, dialysis, or transplant

CVD IN CKD

CVD accounts for 40-50% of all deaths in ESRD patients, with CVD mortality rates approximately 15 times that seen in the general population(4). However, CVD is highly prevalent in patients who progress to ESRD implying that earlier stages of CKD increase the development of CVD. A number of factors have been proposed as risk factors for CVD in CKD including proteinuria, inflammation, anemia, malnutrition, oxidative stress and uremic toxins(5). Ongoing research is investigating whether these (and other) markers may be therapeutic targets. Interestingly, proteinuria correlates with blood pressure, total cholesterol, triglycerides, and inversely correlates with HDL-cholesterol(6). Thus, it remains unclear if proteinuria itself is a risk factor (e.g. a cause of CVD) or a biomarker. Meta-analyses of general population and high risk population cohorts found that both lower eGFR (<60 ml/min/1.73 m²) and higher albuminuria (>10 mg/g creatinine) are predictors of total mortality and CVD mortality; furthermore, eGFR and albuminuria are independent of each other and of traditional CVD risk factors (7, 8). Estimated GFR > 60 ml/min/1.73 m² is not a risk factor for CVD or total mortality.

Dyslipidemia in CKD

Effect of CKD on lipid levels:
CKD is associated with a dyslipidemia comprised of elevated triglycerides and low HDL-cholesterol. Levels of LDL-cholesterol (and thus, total cholesterol) are generally not elevated; however, proteinuria correlates with cholesterol and triglycerides. CKD leads to a down regulation of lipoprotein lipase and the LDL-receptor, and increased triglycerides in CKD are due to delayed catabolism of triglyceride rich lipoproteins, with no differences in production rate(9). CKD is associated with lower levels of apoA-I (due to decreased hepatic expression(10)) and higher apoB/apoA-I. Decreased lecithin-cholesterol acyltransferase (LCAT) activity and increased cholesteryl ester transfer protein (CETP) activity contribute to decreased HDL-cholesterol levels. Beyond decreased HDL cholesterol levels, the HDL in CKD is less effective in its anti-oxidative and anti-inflammatory functions [for review see (11)].

As CKD progresses the dyslipidemia often worsens. In an evaluation of 2001-2010 National Health and Nutrition Examination Survey (NHANES), the prevalence of dyslipidemia increased from 45.5% in CKD stage 1 to 67.8% in CKD stage 4; similarly, the use of lipid lowering agents increased from 18.1% in CKD stage 1 to 44.7% in CKD stage 4(12). Of more than 1000 hemodialysis patients studied only 20% had “normal” lipid levels (defined as LDL<130 mg/dl, HDL > 40 and triglycerides < 150); of 317 peritoneal dialysis patients only 15% had “normal” lipid levels(13). A larger study evaluating dyslipidemia in > 21,000 incident dialysis patients found 82% prevalence of dyslipidemia and suggested a threshold of non-HDL cholesterol > 100 mg/dl (2.6mmol/L) to identify dyslipidemia in CKD stage 5 subjects(14). Peritoneal dialysis is associated with higher cholesterol levels than hemodialysis, although the reasons aren’t fully understood. In subjects who switched from peritoneal dialysis to hemodialysis there was a drop in cholesterol levels of almost 20% following transition (15). The National Kidney Foundation recommends routine screening of all adults and adolescents with CKD using a standard fasting lipid profile (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides), and follows the classification of the National Cholesterol Education Panel for levels (desirable, borderline or high). Although some studies have found associations between Lp(a) and dialysis patients, this is not well defined and there is no current indication for routine screening of Lp(a).

Effect of CKD on lipoprotein composition:

Beyond simply measuring lipid levels, emerging evidence implies that lipoprotein particle size and composition is altered in CKD, with increased small dense LDL and decreased larger LDL particles in CKD subjects compared to controls(16). Small dense LDL is thought to be more atherogenic than larger LDL particles. An emerging theory is that beyond lipid levels or lipoprotein size, lipoprotein particle “cargo” can affect atherosclerosis development and progression. Lipoprotein particles transport numerous bioactive lipids, microRNAs, other small RNAs, proteins, hormones, etc. For example, a recent study compared LDL particle composition between subjects with stage 4/5 CKD and non-CKD controls, and found similar total lipid and cholesterol content, but altered content of various lipid subclasses, for example decreased phosphatidylcholines, sulfatides and ceramides and increased N-acyltaurines(17). Many of these lipid species are known to have either pro- or anti-atherogenic properties and thus could directly affect atherogenesis.

Effect of renal transplantation on lipid levels
:
Dyslipidemia is frequently seen in renal transplant recipients, including increased total cholesterol, LDL-cholesterol, and triglycerides, and decreased HDL-cholesterol. The dyslipidemia may have existed pre-transplant or be related to transplantation associated factors. Cyclosporine increases LDL-cholesterol via both increased production and decreased clearance. Corticosteroids increase both cholesterol and triglyceride levels in a dose-dependent manner. The adverse effects of cyclosporine and corticosteroids on lipid levels appear to be additive(18). Tacrolimus and azathioprine appear to have less induction of dyslipidemia than cyclosporine(19). Sirolimus increases both cholesterol and triglycerides, in part due to decreased LDL-clearance(20).

Effect of nephrotic syndrome on lipid levels:
The dyslipidemia in nephrotic syndrome can be striking with significant elevations of cholesterol, LDL-cholesterol, triglycerides and lipoprotein(a); HDL cholesterol is often low, especially HDL2. The cause of elevated lipid levels is multi-factorial, including reduction in oncotic pressure which stimulates apoB synthesis (although the exact mechanism by which this occurs is not known), decreased metabolism of lipoproteins, and decreased clearance. Patients with nephrotic syndrome have decreased LDL-receptor activity and increased acyl-CoA cholesterol acytransferase (ACAT) and HMG-CoA reductase activity leading to increased LDL-cholesterol levels(21, 22). Low HDL-cholesterol is thought to be due at least in part to LCAT deficiency secondary to accelerated renal loss of LCAT(23). Triglycerides are elevated due to impaired clearance of chylomicrons and triglyceride-rich lipoproteins, as well as increased triglyceride production(24).

Evidence for/against lipid lowering therapy in CKD for CVD outcomes

Given the high prevalence of CVD in CKD, and the robust clinical evidence in non-CKD subjects that lipid lowering reduces CVD outcomes, there is great interest in using lipid lowering therapy in CKD subjects. Statins are the most commonly used lipid-lowering medications and thus far have been shown to reduce CVD events and/or mortality in virtually every population studied. However, CKD patients seem to be a unique population in that at present there is no evidence of benefit for CVD outcomes in dialysis patients with statin therapy. As discussed below it appears that statins can reduce CVD events in pre-end stage CKD subjects, and in post-renal transplant subjects, but not in dialysis patients (Table 2).

Use of statins in pre-ESRD CKD patients:
Although many of the initial statin CVD studies did not include many CKD patients, evidence from sub-group analyses of large statin studies suggested that CKD subjects had similar benefits to non-CKD individuals. For example, the Heart Protection Study (HPS) which assessed >20,000 subjects at high risk of CVD included a subgroup of 1329 subjects with impaired kidney function. In this subgroup those that received simvastatin had a 28% proportional risk reduction and an 11% absolute risk reduction of a major cardiovascular event compared to those randomized to placebo; similar to the effect on the overall cohort(25). Several other studies or meta-analyses similarly predicted that CKD subjects would have reduction in CVD with statin therapy. For example, a meta-analysis of 38 studies with >37,000 participants with CKD but not yet on dialysis found a consistent reduction in major cardiovascular events, all-cause mortality, cardiovascular death and myocardial infarction in statin users compared to placebo groups. There was no clear effect of statin on stroke, nor was there any effect of statin use on progression of the renal disease(26). Thus, for CKD patients with pre-end stage renal disease statins effectively lower total cholesterol and LDL-cholesterol levels and decrease CVD risk. The different statins have different degrees of renal involvement in their metabolism, and providers should be aware of dose restrictions in CKD (Table 3).

Unclear whether to use statins in subjects with nephrotic syndrome:
Several small clinical studies have investigated the use of lipid lowering therapies in nephrotic syndrome, but data is only available for statins and fibrates, and no CVD outcomes data is available. Several small studies using statins have found efficacy in lowering LDL-cholesterol, and that statins were safe and well tolerated(27, 28). Thus, the use of statins in nephrotic syndrome appears to be safe and efficacious in terms of lipid lowering; however, it is not clear if there is any corresponding benefit on either CVD or renal outcomes.

No benefit to statins in subjects with only microalbuminuria:
The Prevention of Renal and Vascular Endstage Disease Intervention Trial (PREVEND IT) randomized 864 subjects with persistent microalbuminuria (urinary albumin of 15-300mg/24h x 2 samples) to fosinopril (an angiotensin converting enzyme inhibitor) or placebo and to pravastatin 20 mg or placebo. Inclusion criteria for the study included blood pressure <160/100 mm Hg and no use of antihypertensive medications and total cholesterol < 300 mg/dl (8 mmol/L) or < 192 mg/dl (5 mmol/L) if patient had known CVD and no use of lipid lowering medications. Although diabetes was not an exclusion criteria, <3% of the subjects had diabetes(29). The use of statin did not affect either urinary albumin excretion or cardiovascular events; however, the use of fosinopril significantly decreased albuminuria and had a trend to reduction in cardiovascular events. Thus, in the absence of other indications for statin therapy, there appears to be no benefit in subjects that solely have microalbuminuria. However, a subsequent analysis found that the subjects with isolated microalbuminuria had an increased risk for CVD events and mortality compared to those without risk factors(30); thus isolated microalbuminuria appears to indicate high risk and further study is needed to determine effective therapies to reduce risk.

No benefit to statins in dialysis patients:

Studies specifically examining the role of statins in ESRD subjects have not found a benefit. The Deutsche Diabetes Dialyse Studie (4D) randomized 1255 type 2 diabetic subjects on maintenance hemodialysis to either 20 mg atorvastatin or placebo daily. The cholesterol and LDL-cholesterol reduction was similar to that seen in non-dialysis patients; however, unlike non-CKD subjects there was no significant reduction in cardiovascular death, nonfatal myocardial infarction or stroke with atorvastatin compared to placebo(31). Similarly, A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis (AURORA) randomized 2776 subjects on maintenance hemodialysis to rosuvastatin 10 mg or placebo. Again, the LDL-cholesterol lowering in dialysis patients was similar to that seen in other studies in non-dialysis patients, but there was no significant effect on the primary endpoint of cardiovascular death, nonfatal myocardial infarction or stroke(32). The Study of Heart and Renal Protection (SHARP) randomized 9270 CKD patients (3023 on dialysis) to simvastatin plus ezetimibe versus placebo. Unlike 4D and AURORA, the SHARP study did report a significant reduction in major atherosclerotic events in the simvastatin plus ezetimibe group, including the dialysis subgroup(33). However, a meta-analysis of 25 studies involving 8289 dialysis patients found no benefit of statin therapy on major cardiovascular events, cardiovascular mortality, all-cause mortality or myocardial infarction, despite efficacious lipid lowering(34). Nevertheless, a post-hoc analysis of the 4D study did demonstrate a benefit of statin therapy in the subgroup that had LDL cholesterol > 145 mg/dl (3.76mmol/l) (35). Although the use of statins in dialysis patients does not clearly cause harm, at present there is no indication for use in dialysis patients, with the exception of a possible benefit in those with significant elevation in LDL-cholesterol.

Why is statin therapy ineffective in dialysis subjects?
Given the robust data demonstrating statin efficacy in CVD risk reduction in virtually all other populations studied, the lack of efficacy in ESRD subjects in perplexing. However, it may be due to different mechanisms of disease progression in ESRD populations compared to other populations. In ESRD subjects there is increased inflammation and oxidative stress as well as increased non-lipid-associated pro-atherogenic factors, which may be the major cause of atherosclerosis development or progression in CKD subjects [for review see (36)]. Therefore, the relative impact of dyslipidemia on CVD development and progression in ESRD subjects may be less than in other CKD and non-CKD subjects, and thus the potential benefit of lipid lowering therapy is reduced. In ESRD subjects with significant hyperlipidemia (such as genetic hyperlipidemias) there may still be a role for statins, or other lipid lowering therapies. Furthermore, while no benefit has been found for statins in dialysis subjects, there is no evidence of increased harm, and thus consideration of lipid lowering medications in particular individuals with ESRD is warranted.

Use of statins in renal transplant recipients:
The Assessment of Lescol in Renal Transplant (ALERT) study randomized 2102 renal transplant recipients to fluvastatin or placebo. There was a non-significant 17% reduction in the combined primary endpoint (cardiac mortality, nonfatal myocardial infarction or coronary intervention procedures) but a significant reduction in cardiac death or myocardial infarction(37, 38). Furthermore, a post hoc analysis suggested that earlier initiation of statins post-transplant was associated with greater benefit(39). However, as with pre-end stage CKD patients there did not appear to be any benefit from statin therapy on progression of renal disease or graft loss in statin treated transplant recipients. (40) Thus, following renal transplant patients should be considered for statin therapy for CVD risk reduction, but not for graft preservation. Several of the statins have drug interactions, particularly with cyclosporine, thus providers must be aware of dose and drug restrictions (Table 3).

Table 2: Use of statins in various CKD subgroups:

Patient population	Statin indicated? Yes/no
Microalbuminuria*	No
CKD 1-4	Yes
Nephrotic syndrome	Unclear
Dialysis patients	No
Renal transplant recipients	Yes

* in the absence of any other indication

Evidence for/against lipid lowering therapy in CKD for renal outcomes

Given the evidence that renal lipid deposition is associated with progression of renal disease itself, there has been an ongoing interest in whether targeting dyslipidemia in CKD can help delay the progression of the renal disease. The dyslipidemia in CKD is associated not only with increased CVD but also with adverse renal prognosis(41, 42). Biopsy studies have found that the amount of renal apoB/apoE is correlated with increased progression of the renal disease itself(43). Animal studies have supported this concept. A meta-analysis of several small, older studies suggested that the rate of decline in GFR was decreased in subjects receiving a lipid-lowering agent (the included studies mainly used statins but the meta-analysis also included a study using gemfibrozil and another using probucol)(44). However, the relationship between lipid levels and renal disease is unclear, as prospective cohort studies have not found any relationship of lipid levels to progression of kidney disease(45). Furthermore, the SHARP study, which included subjects with earlier stages of CKD (stages 3-5 were included) found no benefit of lipid lowering therapy on the progression of renal disease. A meta-analysis of statins in pre-end stage CKD patients found no overall effect of statins on renal disease progression(26) and the ALERT study found no benefit of statin use on renal graft or renal disease parameters(40). Thus, there does not appear to be any use for statins to improve renal function or CKD itself.

Safety of statins in CKD

Statin safety– renal outcomes:
A recent observational study using administrative databases containing information on > 2 million patients suggested that the use of high potency statins was associated with acute kidney injury, especially within the first 120 days of statin use(46). However, a subsequent analysis of 24 placebo controlled statin studies and 2 high versus low-dose statin studies found no evidence of renal injury from statin use(47). These discrepant results can be explained by the quality of the data: in randomized controlled trials, albeit not designed or powered to look at renal injury, data quality tends to be higher than that in administrative data sets, which often contain bias for selection, ascertainment and classification. Furthermore, statins appear to have a nephron-protective role in the prevention of contrast induced acute kidney injury. A meta-analysis of 15 trials examining the effect of statin pre-treatment before coronary angiography found a significant reduction in acute kidney injury in those treated with high dose statin compared to controls treated with either placebo or low dose statin(48). One study specifically examined the use of statins in subjects with diabetes and existing CKD undergoing angiography, and found a benefit to statin pre-treatment in reducing the risk of contrast induced acute renal injury(49). As discussed above, use of statins in pre-end stage CKD or post-renal transplant patients demonstrates neither benefit nor harm on renal outcomes. Thus, based on available evidence there does not seem to be any renal harm from statin use, and the presence of CKD should not be a contra-indication to statin use, although some statins require dose restrictions in CKD (Table 3).

Statin safety – diabetes outcomes:
As a class, emerging evidence demonstrates that statins increase new diagnoses of diabetes(50). As diabetes can lead to or exacerbate renal injury, this is another potential harm of statins. A subsequent meta-analysis of 5 statin trials with >32,000 patients without diabetes at baseline found that high dose statin was associated with increased risk for new diabetes compared to low or moderate dose statin therapy(51). However, the number needed to harm (induce diabetes) is 498 whereas the number needed to treat (prevent cardiovascular events) is 155 for intensive statin therapy; implying that despite the increased risk of new onset diabetes, statin therapy’s benefits outweigh the risks.

Which statins to use in CKD?

The various statins have different degrees of renal clearance; thus, with CKD patients it is important to be aware of the metabolism of the agent of interest and understand if/when dose adjustments are needed. Most statins are primarily metabolized through hepatic pathways, and dose adjustment in early CKD is typically not needed (eGFR> 30 ml/min). However, with more advanced CKD, eGFR< 30 ml/min (or ESRD, although statins are not indicated in this population) most agents have maximum dose restrictions (Table 3).

Table 3: Statin dosing in CKD

Statin	Usual dose range (mg/d)	Clearance route	Dose range for CKD stages1-3	Dose range for CKD stages4-5	Use with cyclosporine
Atorvastatin	10-80	Liver	10-80	10-80	Avoid use with cyclosporine
Fluvastatin	20-80	Liver	20-80	20-40	Max dose 20 mg/d with cyclosporine
Lovastatin	10-80	Liver	10-80	10-20	Avoid use with cyclosporine
Pitavastatin	1-4	Liver/Kidney	1-2	1-2	Avoid use with cyclosporine
Pravastatin	10-80	Liver/Kidney	10-80	10-20	Max dose 20 mg/d when used with cyclosporine
Rosuvastatin	10-40	Liver/Kidney	5-40	5-10	Max dose 5 mg/d with cyclosporine
Simvastatin	5-40	Liver	5-40	5-40	Avoid use with cyclosporine

Beyond statins:

There has been relatively little research into the use of non-statin lipid lowering agents in CKD. There is an emerging interest in niacin in CKD patients for its phosphorus-lowering effects, and niacin has similar lipid-altering efficacy in CKD as opposed to non-CKD subjects. Fibrates are metabolized via the kidney and thus generally contraindicated in CKD. The following sections summarize the available data on the use of other lipid lowering agents in CKD (Table 4).

Niacin:
As niacin is not cleared via the kidney it is theoretically safe in CKD; however, its use is limited due to side effects (predominantly flushing) and a lack of data. Several short term studies have evaluated niacin in CKD patients and it is efficacious in lipid lowering. There is an emerging interest in use of niacin or its analog niacinimide in CKD and ESRD patients for their effects to decrease phosphate levels. A meta-analysis of randomized controlled trials of niacin and niacinamide in dialysis patients found that niacin reduced serum phosphorus but did not change serum calcium levels; furthermore niacin increased HDL levels but had no significant effect on LDL-cholesterol, triglycerides or total cholesterol levels; no CVD outcomes data were provided(52). Animal studies have suggested a beneficial effect of niacin on renal outcomes(53), and clinical literature is suggestive that this may occur in humans. A database study of niacin in veterans reported that niacin may slow progression of chronic kidney disease (reported at the 2014 Kidney Week meeting(54)), but this data is not yet published and further study is needed. Kang et al treated patients with CKD stages 2-4 with niacin 500mg/d x 6 months; niacin led to increased HDL-cholesterol and decreased triglyceride levels, and improved GFR compared to baseline levels(55). Laropiprant has been developed as an inhibitor of prostaglandin-medicated niacin-induced flushing. In a sub-study examining the use of niacin with laropiprant in dyslipidemic subjects with impaired renal function, the use of niacin resulted in a mean decrease in serum phosphorus of 11% with similar effects between those with eGFR above or below 60 ml/min/1.73 m²(56); the parent study reported significant reduction in lipid parameters including a decrease in LDL-cholesterol of 18%, decrease in triglycerides of 25% and an increase in HDL of 20%(57). Thus, there may be an indication for use of niacin in CKD subjects beyond lipid lowering considerations. However, cardiovascular outcome studies evaluating the combination of statin plus niacin have not found any additional benefit compared to statin alone(58, 59); thus, at this time further research is needed in CKD subjects to determine if niacin may be more beneficial than statins, or if the addition of niacin to statin may confer non-CVD benefit, for example, from phosphorus lowering.

Fibrates:
Fibric acid derivatives which are used primarily to raise HDL-cholesterol and lower triglycerides; thus, they target two major components of CKD associated dyslipidemia. However, fibrates are known to decrease renal blood flow and glomerular filtration and they are cleared renally(60); therefore, there is significant concern re their use in CKD. Furthermore, the fibric acid derivatives raise serum creatinine levels and may thus trigger medical investigations into renal disease progression. Thus, there is concern regarding their use in CKD. However, there is a potential for fibric acid derivatives to improve both CVD and CKD outcomes. The acute changes in serum creatinine do not necessarily indicate adverse renal effects. A meta-analysis(61) examined the use of fibrates in CKD subjects and reported beneficial effects to reduce total cholesterol and triglyceride levels and raise HDL-cholesterol levels with no effect on LDL-cholesterol levels. In addition, 3 trials reporting on > 14,000 patients reported that fibrates reduced risk of albuminuria progression in diabetic subjects, with 2 trials (>2,000 patients) reporting albuminuria regression (62-64). This was associated with a reduction in major cardiovascular events, CVD death, stroke and all-cause mortality in subjects with moderate renal dysfunction, but not in those with eGFR > 60 ml/min/1.73m². Thus, despite the elevations in serum creatinine seen with fibrates, there is the potential for both cardiac and renal benefit, and further studies specifically designed to evaluate these outcomes in CKD subjects are urgently needed. At this point, providers are encouraged to consider fibrate therapy for appropriate subjects, especially if statins are not tolerated or are contra-indicated.

Ezetimibe:
Ezetimibe is presently the only member of the class of cholesterol absorption inhibitors. As monotherapy it can lower LDL approximately 15%; however, the majority of research has examined ezetimibe in combination with a statin (primarily simvastatin) where the addition of ezetimibe can induce a further 25% lowering of LDL cholesterol. Ezetimibe is metabolized through intestinal and hepatic metabolism, and does not require any dose adjustment in CKD or ESRD, making it potentially attractive therapy in CKD. Recently, the results of the Improved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE IT) study were presented (American Heart Association Scientific Session November 2014; however, not published as of the time of writing this section) demonstrating that the combination of statin + ezetimibe led to further LDL lowering and improved CVD outcomes compared to statin alone in high risk patients(65). The Study of Heart and Renal Protection (SHARP) compared CVD and renal effects in CKD patients treated with statin + ezetimibe versus placebo. There was a reduction in CVD events(33); however, there was no effect on renal disease progression(66). Note, neither of these studies included an ezetimibe only arm; thus, the effects of ezetimibe monotherapy on outcomes are unknown, although it can be expected to reduce CVD events in proportion to its degree of LDL-cholesterol lowering. A small study evaluating ezetimibe monotherapy in CKD patients found it safe and effective(67). Thus, the use of ezetimibe with or without statin is likely to benefit pre-end stage CKD patients in terms of CVD outcomes (given that the impact of ezetimibe is on lowering LDL-cholesterol we can anticipate lack of CVD benefit in ESRD subjects based on the statin studies and SHARP).

Fish oil:
Omega-3 polyunsaturated fatty acids can lower triglyceride levels, making them a potential therapy in CKD. The role of fish oil/ omega-3 supplements in the general population for prevention of CVD events remains unclear, with some studies suggesting benefit but others finding no CVD protection. A recent meta-analysis found no evidence for CVD protection(68). In CKD patients there is little data and it is conflicting. A small randomized study evaluated omega-3 fish oil supplements, coenzyme Q10, or both in subjects with CKD stage 3 for 8 weeks. The group that received the omega-3 supplements had decreased heart rate and blood pressure and triglycerides, but there was no effect on renal function (eGFR, or albuminuria)(69). Conversely, a study evaluating dietary omega-3 intake found that higher consumption was associated with reduced likelihood of CKD(70). Fish oil supplementation has not been found to have any clear benefit on hemodialysis arteriovenous graft function(71) or on cardiovascular events or mortality in hemodialysis patients(72). Thus, there is no clear benefit to the use of fish oil supplements in CKD, but further research is needed.

Bile acid resins:
The bile acid resins tend to be used less commonly than other classes of lipid lowering agents overall, and their use in CKD is limited by a lack of data. Bile acid resins as a class can lower LDL-cholesterol by 10-20% so they are less effective than statins; furthermore, they can raise triglyceride levels and their use is contra-indicated with elevated triglyceride levels, for example > 400-500 mg/dl (>4.5 – 5.6 mmol/L). Thus, overall bile acid resins are rarely used in CKD patients. However, their metabolism is intestinal and thus there are no required modifications for their use in mild-moderate CKD. Although there are no theoretical concerns regarding their use in ESRD there is no data to address safety or efficacy.

New therapies:
There are a number of new and pending therapies for the treatment of dyslipidemia, including mipomersen (an anti-sense oligonucleotide against apoB-100), lomitapide (a microsomal triglyceride transfer protein inhibitor), evolucumab and alirocumab (monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 [PCSK9]) and evacetrapib and anacetrapib (cholesterol ester transfer protein [CETP] inhibitors). However, to date none of these agents have been studied in CKD; thus, there is no data available to assess their potential use in this population. Mipomersen and lomitapide are both FDA approved for use in homozygous familial hypercholesterolemia; none of the others are yet on the market.

Table 4- Non-statin treatments

Agent	Usual dose range (mg/d)	Clearance route	Dose range for CKD stages1-3	Dose range for CKD stages4-5	Use with cyclosporine
Niaspan	500-2000	Hepatic/renal	No data	No data	No data
Gemfibrozil	1200	Renal	Avoid if creatinine > 2.0 mg/dl	Avoid if creatinine > 2.0 mg/dl	Cautious use
Fenofibrate	40-200	renal	40-60	avoid	Cautious use
Ezetimibe	10	Intestinal/hepatic	10	10	Cautious use
Colsevelam	3750 (6 x 625 mg tablets daily)	Intestinal	No change	unknown	May reduce levels of cyclosporine
Fish oil	4000		No change	Caution	No data

SUMMARY:
CVD is the leading cause of mortality in CKD, and as with the non-CKD population dyslipidemia is a significant contributor. The dyslipidemia of CKD comprises primarily high triglyceride levels and low HDL-cholesterol levels; however, emerging data suggests that the composition of the lipoprotein particles is altered by CKD, and that altered composition and/or lipoprotein cargo may be a cause of the increased CVD in CKD. The use of statins has been shown to be safe and efficacious in lipid lowering in CKD, and of benefit in reducing CVD events in individuals with pre-end stage CKD, or post renal transplant, but not in dialysis patients. The various available agents have different clearance routes, and some statins need dose adjustment in CKD. In patients that cannot tolerate or who have contra-indications to statin therapy, there may be some benefit from use of fibrates or niacin, but further studies are needed to better investigate their use.

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Mori, T. A., V. Burke, I. Puddey, A. Irish, C. A. Cowpland, L. Beilin, G. Dogra, and G. F. Watts. 2009. The effects of [omega]3 fatty acids and coenzyme Q10 on blood pressure and heart rate in chronic kidney disease: a randomized controlled trial. J Hypertens 27: 1863-1872.
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Dietary Treatment of Obesity

1. Introduction

NHANES data reveals that 33% of U.S. adults are overweight, (BMI of 25-29), over 35% are obese (BMI 30 or higher) and over 6% are extremely obese (BMI greater than or equal to 40.0) (1). Obesity is a chronic medical condition requiring long-term therapy (2, 3-5). If left untreated, overweight and obesity can increase the risk degenerative diseases such as diabetes, hypertension, dyslipidemia, coronary artery disease, and metabolic syndrome, and orthopedic problems. In addition, obesity can promote the development of various negative psychological effects, and can diminish one’s quality of life (6).

Self-initiated approaches to weight reduction are often ineffective. We all long for a quick and easy remedy to cure it, when in fact, there is no sure cure. The only effective method to keep off excess weight is through life-long weight management and obesity prevention, involving physical activity, balanced with a healthy diet (3;5). Health professionals can help people become more effective at maintaining a healthy weight, or losing weight when necessary.

Although 1998 NIH guidelines recommend that healthcare professionals advise obese patients to lose weight, the proportion of obese patients who reported being counseled by a healthcare professional has declined since 1994 (7). Yet the fact remains that a modest (10%) weight reduction in obese people is an attainable goal, and often results in clinical improvements of several health-related parameters, even if the individual remains clinically obese (3;8;9). This information should encourage health professionals to advise weight loss for obese patients and that they need not be overwhelmed by their inability to meet excessively ambitious, or unrealistic, weight loss goals (8;10). Smaller amounts of weight loss can still bring considerable health and social benefits.

There is a great deal of misinformation about obesity in many countries today, including the USA. According to a survey by the Natural Marketing Institute, 59% of the general population would benefit from losing weight. Of them, 26% used weight loss products in the past year, 21% used prescriptions, 18% used over the counter medications and 11% used weight loss dietary supplements to maintain and/or manage their weight (11). Only some of these strategies are effective, as we will see in this chapter.

Weight management counseling of overweight and obese patients deserves reconsideration and reemphasis by health professionals because it carries potential for health benefits. Obese patients receiving weight reduction advice from their physicians are significantly more likely to embark on weight loss attempts than those who do not. Yet less than 42% of obese individuals reported that they received weight loss recommendations from their physicians (12). These findings underscore the need for increased health professional involvement in obesity treatment (4;10;13). When physicians are appropriately aware of, and include recommendations for lifestyle changes in counseling their obese patients, results are promising (12;14). Even more importantly, they should stress achievement and maintenance of a healthy weight before obesity becomes apparent.

The 2010 Dietary Guidelines for Americans (Table 1) provides assistance in maintaining and achieving a healthy weight as well as reducing risk of chronic, diet-related diseases through promoting health and a healthy eating pattern (15). MyPlate.gov (Figure 1) and the Dietary Approaches to Stop Hypertension (DASH) recommendations (Table 2) also provide specific guidance on food selection to assure a healthful diet. The 2008 Physical Activity Guidelines for Americans provide science-based guidance to help Americans, ages six and older, improve their health through appropriate forms of physical activity (Table 3). These guidelines are all useful for prevention of weight gain and maintenance of a healthy weight for the weight loss phase of weight control, different physical activity recommendations are involved, and these are discussed in this chapter.

This chapter focuses on steps health professionals can take to help their patients manage their weights more effectively, or to lose weight by dietary means when that is necessary.

Table 1. Dietary Guidelines for Americans, 2010 (15)
Risk Intervention and Goals	Key Recommendations	Special Population Recommendations
Adequate nutrients within calorie needs	Consume a variety of nutrient-dense foods/beverages with the basic food groupsLimit intake of saturated and trans fats, cholesterol, added sugars, salt, and alcohol.Balance intake of calories with energy needs	Adults age >50 should consume vitamin B12 fortified foods or a supplement in crystalline formWomen of childbearing age who may become pregnant should consume foods rich in heme-iron and/or iron-rich plant foods with food rich in vitamin C to enhance absorptionWomen in the first trimester should also consume adequate folic acid via dietary supplement, fortified sources of food containing folic acid as well as other foods naturally high in folic acidOlder adults, people with dark skin, and those not exposed to sufficient sunlight should consume extra vitamin D from vitamin D fortified foods and/or supplements
Weight Management	Maintain body weight in a healthy range by balancing calories with energy expendedPrevent gradual weight gain by making small decreases in food/beverage calories in combination with increases in physical activity	Overweight adults: strive for slow, steady weight loss by decreasing calories and increasing physical activity while maintaining adequate nutrient intakeOverweight children place on a weight-reduction diet only after consultation with a healthcare provider; reduce the rate of weight gain while allowing growth and developmentPregnant women ensure weight gain is appropriate as specified by healthcare provider, since optimal total pregnancy weight gain varies from person to person (14).Breastfeeding women: moderate weight loss is acceptable and does not compromise of the nursing infant’s weight gainOverweight adults and children with chronic diseases and/or on medications: consult a healthcare provider before starting weight reduction to obtain a weight loss plan that ensures other health problems are managed appropriately.
Physical Activity	Engage in regular physical activity and reduce sedentary activities to promote overall health, psychological well-being and a healthy body weightReduce the risk of chronic diseases in adulthood by engaging in at least 30 minutes of moderate-intensity physical activity on most days of the weekGreater health benefits can be obtained by engaging in physical activity that is more vigorous or for a longer durationManage unhealthy body weight gain by engaging in about 60 minutes of moderate-to-vigorous intensity activity on most days per week while not exceeding caloric requirementsSustain weight loss by engaging in at least 60-90 minutes of daily moderate-intensity physical activity while not exceeding caloric requirementsAchieve over-all physical fitness by including a variety of exercises (cardiovascular, stretching, resistance, and calisthenics)	Children and adolescents: engage in at least 60 minutes of physical activity on most, preferably all, days of the weekPregnant women: engage in 30 minutes or more of moderate intensity physical activity most days of the week if no medical or obstetric complications are present; avoid falls and abdominal traumaBreastfeeding women: there are no adverse effects from acute or regular exerciseOlder adults: participate in regular physical activity to help reduce functional declines associated with age
Food Groups to Encourage	Consume a sufficient amount of fruits and vegetables while staying within energy needs; two cups of fruit and 21/2 cups of vegetables per day are recommended for a reference 2,000-calorie intake, with higher or lower amounts depending on the calorie levelChoose a variety of fruits and vegetables each day. In particular, select from all five vegetable subgroups (dark green, orange, legumes, starchy vegetables, and other vegetables) several times a weekConsume 3 or more ounce-equivalents of whole-grain products per day, with the rest of the recommended grains coming from enriched or whole-grain products. In general, at least half the grains should come from whole grainsConsume 3 cups per day of fat-free or low-fat milk or equivalent milk products.	Children and adolescents: consume whole-grain products often; at least half the grains should be whole grainsChildren 2 to 8 years: consume 2 cups per day of fat-free or low-fat milk or equivalent milk productsChildren 9 years of age and older: consume 3 cups per day of fat-free or low-fat milk or equivalent milk products
Fats	Consume less than 10% of calories from saturated fat and less than 300 mg/day of cholesterolLimit intake of fats and oils high in saturated and trans-fatty acids; keep trans-fatty acid intake as low as possibleConsume 20% - 35% of total calories from fat; emphasize polyunsaturated and monounsaturated fatty acids (i.e. fish, nuts, vegetable oils)Choose and prepare meat, poultry, dry beans and milk or milk-products that are lean, low-fat or fat-free	Children ages 2 to 3 : keep total fat intake between 30%-35% of total caloriesChildren and adolescents ages 4 to 18 : consume 25% - 35% of total calories from fat with most fats coming from sources of polyunsaturated and monounsaturated fatty acids (i.e. fish, nuts and vegetable oils)
Carbohydrates	Choose fiber-rich fruits, vegetables, and whole grains oftenChoose and prepare foods and beverages with little added sugars or caloric sweeteners, such as amounts suggested by the USDA Food Guide and the DASH Eating PlanReduce the incidence of dental caries by practicing good oral hygiene and consuming sugar - and starch-containing foods and beverages less frequently
Sodium and Potassium	Consume potassium-rich foods daily such as fruits and vegetables while choosing and preparing foods with little salt	Consume less than 2,300 mg (about 1 tsp) of sodium per day Middle-aged and older adults, African Americans, and people with hypertension aim to consume less than 1,500 mg of sodium per day and meet potassium recommendation of 4,700 mg per day with food.
Alcohol	Those who choose to drink alcohol should do so in moderation; defined as 1 drink per day for women; 2 drinks per day for men	Those engaging in activities that require attention, skill or coordination such as driving or operating machinery: avoid alcohol consumptionThose under the legal drinking age and women who are pregnant should avoid alcohol consumption.
Food Safety	To avoid microbial foodborne illness, clean hands, food contact surfaces, and fruits and vegetablesMeat and poultry should not be washed or rinsedSeparate raw, cooked, and ready-to-eat foods while shopping, preparing, or storing foodsCook foods to a safe temperature to kill microorganismsChill (refrigerate) perishable food promptly and defrost foods properlyAvoid raw (unpasteurized) milk or any products made from unpasteurized milk, raw or partially cooked eggs or foods containing raw eggs, raw or undercooked meat and poultry, unpasteurized juices, and raw sprouts	Infants and young children, pregnant women, older adults, and those who are immunocompromised DO not eat or drink raw (unpasteurized) milk or any products made from unpasteurized milk, raw or partially cooked eggs or foods containing raw eggs, raw or undercooked meat and poultry, raw or undercooked fish or shellfish, unpasteurized juices, and raw sprouts.Pregnant women, older adults, and those who are immunocompromised eat only deli meats and frankfurters that have been reheated to steaming hot.

Table 2. Dietary Approaches to Stop Hypertension (DSH) Diet recommendations (17)
The number of daily servings in food group vary depending on caloric needs.
Food Group	1,200 calories	1,400 calories	1,600 calories	1,800 calories	2,000 calories	2,600 calories	3,100 calories	Serving Sizes
Grains	4-5	5-6	6	6	6-8	10-11	12-13	1 slice bread1 oz dry cereal^b½ cup cooked rice, pasta or cereal ^b
Vegetables	3-4	3-4	3-4	4-5	4-5	5-6	6	1 cup raw leafy vegetable½ cup cut-up raw or cooked vegetable½ cup vegetable juice
Fruits	3-4	4	4	4-5	4-5	5-6	6	1 medium fruit¼ cup dried fruit½ cup fresh, frozen or canned fruit½ cup fruit juice
Fat-free or low-fat milk and milk products	2-3	2-3	2-3	2-3	2-3	3	3-4	1 cup milk or yogurt1½ oz cheese
Lean meats, poultry and fish	3 or less	3-4 or less	3-4 or less	6 or less	6 or less	6 or less	6-9	1 oz cooked meats, poultry or fish1 egg
Nuts, seeds, and legumes	3 per week	3 per week	3 -4 per week	4 per week	4-5 per week	1	1	1/3 cup or 1½ oz nuts2 Tbsp peanut butter2 Tbsp or ½ oz seeds½ cup cooked legumes (dried beans, peas)
Fats and Oils	1	1	2	2-3	2-3	3	4	1 tsp soft margarine1 tsp vegetable oil1 Tbsp mayonnaise1 Tbsp salad dressing
Sweets and sugars	3 or less per week	3 or less per week	3 or less per week	5 or less per week	5 or less per week	Less than 2	Less than 2	1 Tbsp sugar1 Tbsp jelly or jam½ cup sorbet, gelatin dessert1 cup lemonade
Maximum sodium limit^d	2,300 mg/day	2,300 mg/day	2,300 mg/day	2,300 mg/day	2,300 mg/day	2,300 mg/day	2,300 mg/day
Footnotes dietary approaches to Stop Hypertension (DSH) Diet recommendations
Eating patterns from 1,200 to 1,800 calories meet the nutritional needs of children 4 to 8 years old. Patterns from 1,600 to 3,100 calories meet the nutritional needs of children 9 years and older as well as adults. Serving sizes vary between ½ _cup and 1¼ _cups, depending on cereal type. Check product’s Nutrition Facts label.

Table 3. 2008 Physical Activity Guidelines for Americans (18)
Population/Focus Area	Key Guidelines
Children and Adolescents	Children and adolescents” 60 minutes (1 hour) or more of physical activity daily. Aerobic: Most of the 60 or more minutes a day should be either moderate- or vigorous-intensity aerobic physical activity, and should include vigorous-intensity physical activity at least 3 days a week. Muscle-strengthening: As part of their 60 or more minutes of daily physical activity, children and adolescents should include muscle-strengthening physical activity on at least 3 days of the week. Bone-strengthening: As part of their 60 or more minutes of daily physical activity, children and adolescents should include bone-strengthening physical activity on at least 3 days of the week. Encourage young people to participate in physical activities that are appropriate for their age, that are enjoyable, and that offer variety.
Adults	All adults should avoid inactivity. Some physical activity is better than none, and adults who participate in any amount of physical activity gain some health benefits. For substantial health benefits, adults should do at least 150 minutes (2 hours and 30 minutes) a week of moderate-intensity, or 75 minutes (1 hour and 15 minutes) a week of vigorous-intensity aerobic physical activity, or an equivalent combination of moderate- and vigorous intensity aerobic activity. Aerobic activity should be performed in episodes of at least 10 minutes, and preferably, it should be spread throughout the week. For additional and more extensive health benefits, adults should increase their aerobic physical activity to 300 minutes (5 hours) a week of moderate intensity, or 150 minutes a week of vigorous intensity aerobic physical activity, or an equivalent combination of moderate- and vigorous-intensity activity. Additional health benefits are gained by engaging in physical activity beyond this amount. Adults should also do muscle-strengthening activities that are moderate or high intensity and involve all major muscle groups on 2 or more days a week, as these activities provide additional health benefits.
Older Adults	The Key Guidelines for Adults also apply to older adults. In addition, the following Guidelines are for older adults: When older adults cannot complete 150 minutes of moderate-intensity aerobic activity a week because of chronic conditions, they should be as physically active as their abilities and conditions allow. Older adults should include exercises that maintain or improve balance if they are at risk of falling. Older adults should determine their level of effort for physical activity relative to their level of fitness. Older adults with chronic conditions should understand whether and how their conditions affect their ability to do regular physical activity safely.
Safe Physical Activity	To perform physical activity safely and reduce the risk of injuries and other adverse events, people should: Understand the risks but remember that physical activity is safe for almost everyone. Choose types of physical activity that are appropriate for their current fitness level and health goals, because some activities are safer than others. Increase physical activity gradually over time when more activity is necessary to meet guidelines or health goals. Inactive people should “start low and go slow,” gradually increasing how often and how long activities are done. Protection: using appropriate gear and sports equipment, looking for safe environments, following rules and policies, and making sensible choices about when, where, and how to be active. Those with chronic conditions or symptoms should be under the care of a health-care provider and should consult the health-care provider about the types and amounts of activity appropriate for them.
Women During Pregnancy and the Postpartum Period	Healthy women who are not already highly active or doing vigorous-intensity activity should complete at least 150 minutes of moderate-intensity aerobic activity a week during pregnancy and the postpartum period. Preferably, this activity should be spread throughout the week. Pregnant women who habitually engage in vigorous-intensity aerobic activity or who are highly active can continue physical activity during pregnancy and the postpartum period, provided that they remain healthy and discuss with their health-care provider how and when activity should be adjusted over time.
Adults with Disabilities	Adults with disabilities, who are able to do so, should complete at least 150 minutes a week of moderate-intensity, or 75 minutes a week of vigorous-intensity aerobic activity, or an equivalent combination of moderate- and vigorous-intensity aerobic activity. Aerobic activity should be performed in episodes of at least 10 minutes, and preferably, it should be spread throughout the week. Adults with disabilities, who are able to do so should also do muscle-strengthening activities of moderate or high intensity that involve all major muscle groups on 2 or more days a week, as these activities provide additional health benefits. When adults with disabilities are not able to meet the Guidelines, they should engage in regular physical activity according to their abilities and should avoid inactivity. Adults with disabilities should consult their health-care providers about the amounts and types of physical activity that are appropriate for their abilities.
Chronic Medical Conditions	Adults with chronic conditions can obtain important health benefits from regular physical activity. When adults with chronic conditions perform activity according to their abilities, physical activity is safe. Adults with chronic conditions should be under the care of a health-care provider. People with chronic conditions and symptoms should consult their health-care provider about the types and amounts of activity appropriate for them.

2. RATIONALE FOR DIETARY TREATMENT

Over two-thirds of adults in the United States are currently overweight or obese (19). The percent of overweight and obese people has risen to this point over the past several decades, among men and women, all ethnic groups, all ages, and all education levels. From 1960 to 2010, the prevalence of obesity in the United States more than doubled, going from 13.4% to 36.1% in adults ages 20 to 74, although the prevalence of overweight remained relatively stable (20). In recent years the trend has begun to level off, with one large study finding no significant differences in the incidence of obesity in adults between 2003-2004 and 2011-2012 (21). However, the incidence of both overweight and obesity remain high, and with the medical costs for an obese person averaging $1,429 more per year (in 2008 dollars) than a normal weight person, attention to weight control and maintenance remains paramount for medical practitioners (22).

Weight control has health advantages, and therefore maintaining or achieving a healthy weight is important for all Americans. Obesity is associated with an increase in mortality rates. Obese individuals have an increased risk of death of at least 20% for all-cause and CVD associated mortality (23). Excess weight might contribute to as much as 41% of uterine cancers, and 10% of gallbladder, kidney, liver, and colon cancers (. In weight control prevention it is especially paramount because once weight and adiposity have surpassed healthy levels, they are difficult to reduce. Therefore, it is important for health professionals to monitor the weights of all their patients and to provide anticipatory guidance so that those who are already at healthy weights remain so. The 2010 Dietary Guidelines for Americans (Table 1) stress maintenance of a body weight within a healthy range by balancing calories from foods and beverages with calories expended, by preventing gradual weight gain over time, by making small decreases in foods and beverages, and by increasing physical activity. However, this is easier said than done. The chapter will assist health professionals in operationalizing these recommendations.

3. EVALUATING OVERWEIGHT AND OBESITY

This section outlines a stepwise approach for assessing overweight and obesity.

3.1 Assess Body Fat Burden and Health Status

Before any patient is placed on a reducing diet, where caloric intake is greatly reduced, often alongside increases in energy expenditure, medical assessment of weight, fat distribution, and health risks is essential.

3.2 Measure Body Mass Index (BMI) as an Indirect Measure of Body Fat Burden

Weight should be measured, without clothing, on electronic scales, which provide accurate weights even for heavy patients. Scales should be calibrated to ensure accuracy. Height is best measured with a wall-mounted stadiometer or against a wall rather than on beam-balance scales, which are unsteady and unreliable. Body fat is difficult to measure directly and accurately in clinical practice. Therefore, body mass index (BMI), which is highly correlated with total body fat and future health risks, is recommended as the best surrogate method of capturing body fat, although it can overestimate body fat in individuals with high muscle mass. BMI can be calculated using the following formulas (24):

BMI= (weight lbs ÷ height inches² ) x 703 or BMI = weight kilograms ÷ height meters² (24)

Table 4 presents the National Institutes of Health (NIH) classification of BMI values for adults (24). These values are based on abundant data associating higher BMI levels with higher health risks. Although individuals with the same BMI often differ somewhat in the amount of body fat they have, this is still a useful approximation that can be performed quickly and inexpensively in clinical settings.

Table 4. Classification of Weight Status by Body Mass Index (BMI)
Classification	BMI ( kilogram/m ² )
Underweight	<18.5
Normal weight	18.5-24.9
Overweight	25-29.9
Obesity Class 1	30-34.9
Obesity Class 2	35-39.9
Extreme Obesity Class 3	>40

Individuals with a BMI under 18.5 are classified as underweight, whereas those with a BMI over 25 are considered overweight; those over BMI 30 are classified as class 1 obesity, those over BMI 35 as class 2 obesity, and those over BMI 40 as extreme, or class 3, obesity. In general, the orthopedic and metabolic hazards increase with increasing BMI. Tracking changes in BMI, as well as body weight itself, are easy to use tools for monitoring body composition over time, identifying those at risk for developing overweight and obesity, and monitoring the success of those undergoing weight loss therapy.

The distribution of fat on the body, as well as its sheer amount, also alters risk of some metabolic disorders. The reasons for this are becoming clear as the role of adipose tissue as an endocrine organ is more fully understood. Excess abdominal fat in the viscera, characterized by an accumulation of fat centrally (sometimes referred to as android "apple" or abdominal fat distribution or ectopic fat) is associated with greater risk of certain chronic degenerative diseases than is a peripheral fat deposition pattern (gynoid "pear" or lower body fat pattern).

Although the causal associations between certain diseases and body fat distribution are still a matter of debate (28;27), measuring waist circumference in addition to BMI is still clinically useful in assessing risk posed by body fat distribution (24;5;28-31).

Visceral and subcutaneous fat are difficult to measure accurately in office practice. Waist circumference, taken at the level of the umbilicus (belly button) with a plastic or other type of non-stretchable measuring tape, is a reasonable proxy for assessing the likely size of visceral fat deposits and the extent of abdominal obesity. Waist circumference is easier to measure and more straightforward to interpret than are waist-to-hip ratios. It is generally used as the standard in assessing central vs. peripheral fatness. The cut-points for increased risk are a waist circumference of greater than 35 inches (>88cm) in women, or greater than 40 inches (>102cm) in men (24; 31). Although the usefulness of these absolute cut-offs have been questioned due to the many possible confounding variables in their relationship with health, monitoring changes over time is advocated (32). Measuring waist circumference is most useful for defining risk in obese patients with BMI 25-35 kg/m2. Obese patients with BMIs over 35 kg/m2 already have elevated risk, so waist circumference measurements may be less necessary for them (31).

Table 5 shows how risks of weight related conditions such as type 2 diabetes, hypertension, and cardiovascular disease increase with greater BMI and waist circumference. Patients with high waist circumference may need increased monitoring and treatment of blood pressure, unhealthy blood lipid profiles, and other cardiovascular risk factors. Physical inactivity and smoking increase health risk still further. They act synergistically and apparently increase the severity of the other risk factors present as well as increasing risks themselves in other ways. Elevated serum triglycerides and lower HDL are other markers for increased cardiovascular risk that increase with high waist circumference.

Table 5. Classification of Risk of Type 2 Diabetes, Hypertension and Cardiovascular Disease Associated with Weight
Classification of Fatness Status by BMI and Waist Circumference	Increase in Disease Risk for Type 2 Diabetes, Hypertension and Cardiovascular Disease Over Normal Weight and Waist Circumference
	Waist circumference Women <35 inches Men < 40 inches	Waist circumference Women >35 inches Men > 40 inches
Underweight (BMI <18.5)	---	---
Normal (BMI (18.5-24.9)	---	---
Overweight (BMI 25-29.9)	Increased	High
Obese Class 1 (BMI 30-34.9)	High	Very high
Obese Class 2 (BMI 35-39.9)	Very high	Very high
Extreme Obesity Class 3 (BMI >40)	Extremely high	Extremely high

3.4 Document Other Risk Factors and Comorbidities That Increase Risk and Have Other Implications for Therapy

The presence of risk factors or already clinically apparent diseases further increases the health risk of obesity over that evident with high BMI and high waist circumference alone. Table 6 describes different conditions that further add to the adverse health effects of overweight and obesity itself (24). Weight loss can help lower elevated blood pressure, blood glucose, both total and low-density lipoprotein levels (LDL), plasma cholesterol and triglyceride levels, and raise high density lipoprotein (HDL) cholesterol levels in those with abnormally high values. Other modalities of treatment, including pharmacologic therapy, may also be necessary to bring some patients into healthy ranges.

Table 6. Risk Factors and Comorbidities that Increase the Risks of Morbidity from Overweight
Level of Risk	Conditions
High Absolute Risk	Established coronary heart disease or other atherosclerotic diseaseType 2 diabetesSleep Apnea
High absolute Risk if 3 or More of These Risk Factors are Present	HypertensionCigarette smokingHigh low-density lipoprotein cholesterolLow high density lipoprotein cholesterolImpaired fasting glucoseFamily history of early cardiovascular diseaseAge: >45 in men or >55 in women
Increased Risk	Increased surgical riskPsychological disorders such as depressionOsteoarthritisHirsutism (presence of excess body and facial hairGallstonesStress incontinenceGynecologic problems such as amenorrhea and menorrhagia

3.5 Determine if the Patient is a Candidate for Weight Loss

All individuals with a BMI over 30, and those with a BMI between 25-29.9 with a high waist circumference or one or more of the risk factors listed in Table 6, are potential candidates for weight reduction. Patients who have a BMI between 25-29.9, but who do not have any risk factors or comorbidities should be counseled to avoid further weight gain (23). The goal of weight control is both the reduction of weight and the maintenance of healthy body weight over the long term. Weight loss should be achieved through a high-intensity lifestyle intervention, which is discussed further in section 4.2, if possible (23). If the patient is not open to weight loss, at least prevention of further weight gain should be attempted. Those with very high BMIs (over 35) are unlikely to be able to achieve sufficient fat loss on a usual low calorie diet of 1,200 to 1,500 calories without regimes that must continue for many months. They should be referred for care to a multidisciplinary team specializing in obesity for treatment with very low calorie diets, and possibly pharmacology or surgery (23).
Some individuals whose weights are at healthy levels and who are without weight associated health problems also may wish to lose weight. These patients need to have their concerns about diet addressed, but should not embark on reducing diets since there are no medical indications for them to do so. They should encouraged to maintain their weight within a healthy range, and counseled to follow dietary recommendations from ChooseMyPlate.gov (Figure 1), the Dietary Approaches to Stop Hypertension (DASH) eating plan (Table 2), or the Dietary Guidelines for Americans (Table 1).

Figure 1. ChooseMyPlate.gov (F)

4. Choose Treatment Options

The following section covers the various means of treating obesity, including dietary changes, medications, and/or surgical options. Diet plays a critical role in all of these options.

4.1 Assess the Patient’s Readiness and Willingness to Lose Weight

The previous sections provide the rationale for assessment of the health risks associated with obesity, the potential health benefits accruing from weight loss, and the importance of then maintaining a healthy body weight. Weight control requires behavioral change, which cannot happen without patient buy-in to the process. Therefore, the health risks of overweight and obesity need to be communicated, and patient readiness to change needs to be established. Table 7 outlines the various stages of behavior change as conceptualized by Kushner based on Prochaska’s model of behavior change, often referred to as the Transtheoretical Model of Behavior Change (6). It is important to note many patients will not progress through the outlined stages linearly, but rather will go back and forth repeatedly among stages. Therefore, timing is important and the clinician must watch for an appropriate time to bring up or follow through on the issue.

Table 7. Transtheoretical Model of Behavior Change (5)
Stage	Characteristics	Patient Verbal Cues
Pre-contemplation	Unaware of problem, no interest in change	“I’m not really interested in weight loss. It’s not a problem.”
Contemplation	Aware of the problem, beginning to think of changing	“I need to lose weight but with all that’s going on in my life right now, I’m not sure if I can.”
Preparation	Realizes benefits of making changes and thinking about how to make change	“I have to lose weight, and I’m planning to do that.”
Action	Actively taking steps toward achieving the behavioral goal, but only for a brief period (less than 6 months)	“I’m doing my best. This is harder than I thought.”
Maintenance	Initial treatment and behavioral goals reached and sustained for a longer period of time (e.g., more than 6 months)	“I’ve learned a lot through this process.”

Often, those who are at highest health risk due to obesity are unaware of how serious their weight-related problems are, or are in deep denial about them. The consequences of excess weight, including long-term implications, must therefore be raised and carefully explained. Helping patients to draw connections between the short-and long-term health consequences of their current weight, and the implications this will have on things they care about, such as their family or the ability to participate in activities they enjoy, may aid in empowering patients to progress through the various stages of behavior change.

Once patient readiness and willingness to lose weight has been established, a plan of attack needs to be jointly devised with the patient. Some patients are ready to start a treatment program immediately, and the patient and counselor are able to begin setting goals together right away. Other patients have reservations or other issues keeping them from reaching the action stage needed to embark upon their weight loss goals, making it important for the counselor to address these road-blocks before moving on. For patients who are not ready to act, the issue should be deferred and brought up again at the next visit, rather than dropping the subject entirely. Some groups of patients are unable or unwilling to embark on a weight reduction program at all. Even patients who are unwilling to embark on a reducing diets may be willing to take steps to avoid further weight gain, or may be willing to work on other risk factors such as smoking cessation or increasing physical activity. These activities should be encouraged. For those who are ready and raring to go, a referral to a registered dietitian should be provided where the subject can be addressed in-depth.

4.2 Decide if Dietary Treatment is the Appropriate Option

Weight reduction with dietary treatment is in order for virtually all patients with a BMI over 30, as well as those with a BMI of 25-29.9 with comorbidities. A dietary approach to weight loss should be executed in the context of comprehensive lifestyle intervention whenever possible. This type of intervention involves frequent, in-person encounters with a trained interventionist in an individual or group setting, and incorporates a moderately reduced calorie diet, increases in physical activity, and the use of behavioral techniques to facilitate adherence to recommendations. The gold standard is a comprehensive, high-intensity, on-site program with greater than 14 sessions in 6 months, provided either in a group or individually, by a trained interventionist, and lasting for at least 1 year. When a comprehensive lifestyle intervention is not feasible, other dietary-based approaches, such as electronically based programs and commercial programs, which will be discussed in further detail later, can be appropriate alternatives (23).

For some patients, however, a low calorie (hypocaloric) diet alone may not be enough to prompt significant and lasting weight loss (34).For patients who have failed to lose on a comprehensive lifestyle program, for those with a BMI greater than 30, or greater than 27 if one or more comorbidities are present, and who are likely to have little success with a purely dietary approach on the basis of a history of many failures, other steps may be in order. This is especially important for those with class 2 (BMI>35) and 3 (BMI > 40) obesity, referral to a multidisciplinary obesity treatment team for adjunctive therapies (i.e., very low calorie diets, pharmacological treatment, and/or gastric bypass surgery) is warranted (23).

4.3 Decide if Drugs will be Useful Adjunctive Therapy to the Reducing Diet

Prescription drugs are one form of adjunctive therapy that may be considered for those with a BMI greater than 30, or a BMI of 27 and above if one or more comorbidities are present, who are unable to lose weight with dietary measures alone. Weight loss drugs are only adjuncts to, rather than substitutes for, reducing diets, however, and a reducing diet will still be necessary. Without a hypocaloric diet, drugs are unlikely to be effective. The addition of weight loss medication to a dietary-based weight loss regimen can help patients lose up to 10% of their initial body weight, with most weight loss occurring in the first six months (35). Table 8 provides an overview of prescription medications that are available. Note that none are totally free of side effects.

Table 8. Prescription Medications Available in the United States for Weight Loss (26)
Generic Name + (Trade Name)	Food and Drug Administration Approval for Weight Loss	Drug Type	Common Side Effects
Orlistat (Prescription: Xenical^{^TM)}*OTC Brand: Alli^TM	Yes; long term for adults and children age 12 and older *Alli^TM for adults only	Lipase Inhibitor	Gastrointestinal issues (cramping, diarrhea, oily spotting) Do not take with cyclosporine
Lorcaserin (Belviq^TM)	Yes; long term for adults	Serotonin Receptor Antagonist	Headache, dizziness, nausea, fatigue, dry mouth Do not take with Selective Serotonin Reuptake Inhibitors (SSRIs) or Monoamine Oxidase Inhibitors (MAOIs)
Phentermine-Topiramate (Qsymia^TM)	Yes; long term for adults Contrindicated in women who are pregnant or may become pregnant	Appetite Suppressant/Seizure Treatment	Tingling of hands and feet, trouble sleeping, taste alterations, dry mouth constipation, dizziness, birth defects
Bupropion – Naltrexone (Contrave^TM)	Yes; long term for adults	Depression Treatment/Alcohol and Opioid Abuse Treatment	Nausea, constipation, headache, vomiting, dizziness, insomnia, dry mouth, diarrhea, increased blood pressure and heart rate, seizures, suicidal thoughts and behaviors
Phentermine (Adipex-P^TM, Suprenza^TM, Zantryl^TM)	Yes; short term (up to 12 weeks) for adults	Appetite Suppressant	Increased blood pressure and heart rate, sleeplessness, nervousness
Diethylpropion (Tenuate^TM)	Yes; short term (up to 12 weeks) for adults	Appetite Suppressant	Dizziness, headache, sleeplessness, nervousness
Phendimetrazine (Bontril PDM^TM, Adipost^TM, Melfiat^TM)	Yes; short term (up to 12 weeks) for adults	Appetite Suppressant	Sleeplessness, nervousness
Benzphetamine (Didrex^TM)	Yes; short term (up to 12 weeks) for adults	Appetite Suppressant	Restlessness, anxiety, sleeplessness, headache
Bupropion (Wellbutrin^TM)	No	Depression Treatment	Dry mouth, insomnia
Topiramate (Topamax^TM)	No	Seizure Treatment	Numbness of skin, change in taste
Zonisamide (Zonegran^TM)	No	Seizure Treatment	Drowsiness, dry mouth, dizziness, headache, nausea
Metformin (Glucophage^TM)	No	Diabetes Treatment	Weakness, dizziness, metallic taste, nausea
Byetta (Exenatide^TM, Bydureon^TM)	No	Diabetes Treatment	Nausea

Many of the Food and Drug Administration (FDA)-approved weight-loss medications are approved only for short-term use (short term is usually interpreted to mean use up to 12 weeks), although some physicians still prescribe them for longer periods of time (35). Only four prescription drugs are currently approved for long-term use in weight reduction: Orlistat (Xenical^TM), Lorcaserin (Belviq^TM), Phentermine-Topiramate (Qsymia^TM), and Bupropion–Naltrexone (Contrave^TM).

Orlistat is available for both prescription (Xenical^TM) and over-the-counter at a lower dose as Alli^TM. Over-the-counter Alli^TM is available only to adults aged 18 and older, and is a half-dose version of prescription Orlistat (http://www.myalli.com) (35). Orlistat operates at the level of the gut to inhibit pancreatic lipase, blocking the absorption of about one third of fat consumed. Use over one to two years can lead to a weight loss of five to seven pounds (35). Adherence to a reduced calorie diet with less than 30% calories from fat is necessary while on either Orlistat or Ali. Both Orlistat and Ali’s disadvantages include fat malabsorption, sometimes accompanied by anal leakage, and decreased absorption of fat-soluble vitamins. Because of this decrease in fat-soluble vitamin absorption, patients taking either version of the drug should be advised to take a multivitamin supplement containing fat-soluble vitamins to ensure adequate nutritional status (35). Dietetic counseling is helpful in managing weight loss.

Lorcaserin (Belvique^TM) is another weight loss drug approved for long term use and is available by prescription only. Studies evaluating its effectiveness found that 47% of those who used the drug lost at least 5% of their initial body weight (35). Qsymia^TM is another long-term weight loss drug that was approved by the FDA in 2012. Qsymia^TM is a combination of an appetite suppressant, phentermine, and a seizure medication, topiramate. Studies found that after 1 year using the recommended dose of the drug, 62% of patients lost greater than 5% of their initial body weight (35). ContraveTM is the newest long-term drug to treat obesity, and was approved in September 2014. ContraveTM is a combination of bupropion, an antidepressant, and naltrexone, a medication used to treat alcohol and opioid dependence. Studies showed that after 1 year, 42% of the non-diabetic patients tested lost at least 5% of their initial body weight. (36). With all long-term weight loss drugs, if at least 5% of initial body weight is not lost by 12 weeks, use of the drug should be discontinued as it is unlikely to be effective later, and therefore the risks outweigh the putative benefits (35).

Phentermine (Suprenza^TM), phendimetrazine (Adipost^TM), diethylpropion (Tenuate^TM), and benzphetamine (Didrex^TM) are modestly effective prescribed anorectic agents approved for short-term use (12 weeks in a 12 month period) by the Food and Drug Administration (FDA) (35). Phentermine and diethylpropion are widely prescribed, as they are relatively inexpensive (approximately $30 for a one-month supply), and provide slight stimulatory effects. However, little research has been done on their long-term side-effects (39).

The off-label use of bupropion (Wellbutrin^TM), a drug originally approved by the FDA for aiding in smoking cessation, has become popular in the past few years for weight control. Bupropion enhances norepinephrine and weakly blocks dopamine reuptake and is being studied for the treatment of obesity. Bupropion could be considered if a patient presenting with obesity wanted to quit smoking as well and lose weight (38). Short term side effects most often reported are agitation, dry mouth, insomnia, headache, nausea, constipation, and tremor. However, its long-term effects on weight loss are not clear, and its use must be accompanied by a low-calorie diet if it is to help in weight loss.

Topiramate (Topamax^TM) and zonisamide (Zonegran^TM) are anticonvulsants that were originally approved to treat epilepsy. They are also sometimes used off-label for their weight-loss effects. However, adverse effects have also been reported, most commonly difficulty with memory, parathesia, difficulty concentrating, and mood problems. These drugs are approved by the FDA for epilepsy only, and not for weight loss (38).

Metformin (Glucophage^TM) is a diabetes medication that may promote small amounts of weight loss in people with obesity and type 2 diabetes. One study found that patients treated with metformin for diabetes lost 2kg more at 6 months compared to placebo, and maintained at 1 kg at 4 years follow-up. It is unclear, however, if weight loss on metformin is related to improved glucose tolerance or the drug itself (40).

Byetta^TM (exenatide) and pramlintide are sometimes used in treating the comorbidities of obesity. Both compounds affect the gastrointestinal hormones that regulate glucose homeostasis, gastric emptying, and satiety. Exenatide (Byetta^TM) is used as an adjunctive therapy for improving glycemic control in patients with type 2 diabetes who also take metformin or sulfonylurea. Pramlintide is an adjunctive therapy for patients with type 1 or type 2 diabetes who use insulin at mealtimes. Usually patients with diabetes gain weight with better glucose control, however, with these drugs, better blood glucose control is often associated with weight loss, at least in preliminary studies. The most common side effect of these medications is nausea (38).

Major disappointments have resulted as research on the once promising class of drugs known as cannabinoid (CB1) receptor antagonists has continued. Rimonabant (Acomplia^TM) was the first CB1 receptor blocker approved for use in the world. Its suggested use was for patients with a BMI of 30 or more, in conjunction with exercise and diet, to aid in weight loss. CB1 receptors are located in the brain, gastrointestinal tract, adipose tissue, heart, pituitary, and adrenal glands, and if they are stimulated, these receptors increase appetite. Blockage of these receptors is thought to decrease appetite. However, the FDA ruled that Rimonabant carried too much risk to be approved for use in the United States, with side effects including nausea, anxiety, diarrhea, and depressed mood that, in severe cases, led to suicide (38). In 2009, the European Medicines Agency (EMEA) also concluded that the benefits of Rimonabant no longer outweighed the risks, and marketing authorization for the drug in the European Union was officially revoked (43). Investigation into the cannabinoid (CB1) receptor antagonist class of drugs has since ceased (39).

Other areas of research for future weight loss drugs include drugs combining appetite suppressants and those that affect addiction, drugs affecting gut hormones that influence appetite, drugs that work to shrink the blood vessels supplying fat cells, drugs targeting genes associated with obesity, and manipulation of gut bacteria (35).

Sibutramine (Meridia^TM) was a commonly used obesity drug first introduced in 1997. However, it was voluntarily withdrewn from US markets by its manufacturer in 2010 after clinical trial data indicated that the drug increased the risk for heart attack and stroke. It should not be prescribed or used for the treatment of obesity (44).

Some of the surprisingly positive effects with weight loss drugs are due the fact that the medications are not what they seem to be but rather adulterated and contain undeclared drugs. The FDA releases an extensive list of tainted weight loss products, many of which contain undeclared drugs (Table 9) (44). If patients are taking any of these contaminated products, they should be advised to stop immediately. Table 9 provides a comprehensive list of these tainted products on the market from 2011 onwards, along with the undeclared pharmaceutical/chemical included in the product. There are also other drugs that may be added although they are no longer available for distribution through legitimate sources because of adverse and sometimes fatal side effects including Fen-Phen^TM, Redux^TM, Pondimen^TM, fenfluramine, Meridia^TM, and dexfenfluramine.

Table 9. FDA’s List of Tainted Weight Loss Products (33) The Undeclared Drug/Chemical Ingredient is Listed After Each Product in Parentheses
1 Day Diet (Sibutramine)	7 Days Herbal Slim (Sibutramine)	24 Ince (Sibutramine)
A-Slim 100% Natural Slimming Capsule (Sibutramine)	Acai Berry Soft Gel ABC (Sibutramine)	Advanced (Sibutramine)
Advanced Blue (Sibutramine)	Advanced Slim 5 (SIbutramine)	Asset Bee Pollen (Sibutramine)
Asset Bold (Sibutramine)	Asset Extreme (Sibutramine)	Asset Extreme Plus (SIbutramine)
B-Perfect (Sibutramine)	Be Inspired (Sibutramine)	Beautiful Slim Body (Sibutramine)
Bella Vi Insane Amp’d/Bella Vi Amp’d Up (Sibutramine)	Best Line Suplemento Alimenticio (Sibutramine)	Best Share Green Coffee: Brazilian Slimming Coffee (Sibutramine)
Bethel 30 (Sibutramine)	Body Beauty 5 Days Slimming Coffee (Sibutramine)	Botanical Slimming Soft Gel (Sibutramine)
Burn 7 (Sibutramine)	Celerite Slimming Capsules (Sibutramine)	Citrus Fit Gold (SIbutramine)
DaiDaiHuaJiaoNang (Sibutramine and Phenolphthalein)	Diet Master (Sibutramine)	Dr. Mao Slimming Capsules (Sibutramine)
Dr. Ming’s Chinese Capsule (Sibutramine)	Dream Body Slimming Capsule (Sibutramine)	Extreme Body Slim (Sibutramine)
Fat Zero (Sibutramine and Phenolphthalein)	Fruit & Plant Slimming (Sibutramine)	Fruit Plant Lossing Fat Capsule (Sibutramine)
Goodliness Fat-Reducing Capsules (Sibutramine)	Hot Detox (Sibutramine)	Infinity (Sibutramine)
Instant Slim/ Shou Fu Ti Tun Guo Xiang Xing Jian Fei Jiao Nang (Sibutramine)	Ja Dera 100% Natural Weight Loss Supplement (Sibutramine)	Japan Hokkaido Slimming Weight Loss Pills (Sibutramine, Benzocaine, Phenolphthalein and Diclofenac)
Japan Rapid Weight Loss Diet Pills Green (Phenolphthalein)	Japan Rapid Weight Loss Diet Pills Yellow (Sibutramine and Phenolphthalein)	Japan Weight Loss Blue (Sibutramine, Analogs of Sibutramine, and Ephedrine Alkaloids)
Jimpness Beauty Fat Loss Capsules (Sibutramine)	La Jiao Shou Shen (Sibutramine)	Leisure 18 Slimming Coffee (Sibutramine)
Lingzhi Cleansed Slim Tea (Sibutramine)	Lipo 8 Burn Slim (Sibutramine)	Lishou (Sibutramine)
Lite Fit USA (SIbutramine)	Lose Weight Coffee (Sibutramine)	LX1 (DMAA)
Magic Slim (Sibutramine)	Magic Slim Tea (Sibutramine)	Magic Slim Weight Reduction Capsule (Sibutramine)
MAXILOSS Weight Advanced (Sibutramine)	MAXILOSS Weight Advanced Blue (Sibutramine)	Meizi Evolution (Sibutramine)
Meizitang Citrus (Sibutramine)	Mix Fruit Slimming (Sibutramine)	Natural Body Solution (Sibutramine)
New You (Phenolphthalein)	P57 Hoodia (Sibutramine)	Pai You Guo Slim Tea (Sibutramine and Phenolphthalein)
Paiyouji Plus (Sibutramine)	Perfect Body Solutions (Sibutramine)	PhentraBurn Slimming Capsules (Sibutramine)
Sheng Yuan Fang (Sibutramine)	Slender Slim 11 (Sibutramine)	Slim Forte Slimming Capsule/Slim Forte Double Power Slimming Capsule (Sibutramine)
Slim Forte Slimming Coffee (Sibutramine)	Slim Max (Sibutramine)	Slim Trim U (Sibutramine)
Slim Xtreme Herbal Slimming Capsule (Sibutramine)	SLIMDIA Revolution (SIbutramine)	SlimEasy Herbs Capsule (Sibutramine)
SlimExtra Herbal Capsule (Sibutramine)	Slimming Diet (Sibutramine)	Slimming Diet Berry Plus (Sibutramine)
Strawberry Balance (Sibutramine)	Super Slim (Sibutramine)	Super Slimming (Sibutramine)
Sport Burner (Fluoxetine)	Tengda (Sibutramine)	Thinogenics (SIbutramine)
Toxin Discharged Tea (Fluoxetine)	Trim-Fast Slimming Softgel (Sibutramine)	Ultimate Formula Bee Pollen Capsules (Sibutramine)
Vitaccino Coffee (Sibutramine)	XIYOUJI QINGZHI CAPSULE (Sibutramine)	Zi Xiu Tang Bee Pollen Capsules (Sibutramine)

Drugs for weight loss are of limited efficacy, some patients cannot afford them, and all of them have side effects. About one fourth of all individuals who are prescribed medications will not have the expected response (39). Patients who are likely to respond to drugs tend to do so within the first month of therapy. If they fail to lose four pounds (1.8 kilograms) in the first four weeks, the drug is unlikely to be effective, and it may be appropriate to discontinue its use. A loss of four pounds within the first four weeks generally predicts weight loss of at least 5% body weight by six months of therapy, if the diet and drug continue to be used (39).

Dietary supplements purported to be helpful in weight loss are discussed in section 9.7.3 (Dietary Supplements and Weight Loss). No supplement currently on the market is both safe and effective for weight loss.

4.4 Rule Surgical Options In or Out

Surgical options such as adjustable gastric banding, or more invasive techniques such as rou en y gastric bypass,, sleeve gastrectomy or biliopancreatic diversion with duodenal switch surgery are recommended only for patients classified as Obesity class 2 or above (BMI>35), or as Obesity class 1 (BMI >30) with comorbidities (23). Patients who opt for the surgical route must adhere to certain dietary recommendations before the surgery is performed to show they are able to follow a hypocaloric diet. After surgery, food intake is altered and meals must be smaller because gastric capacity is considerably limited (46). Patients will be required to adhere to a strict, multi-stage diet post-surgery to heal and adjust to new gut physiology short term, as well as to promote weight loss long-term. A post-operative weight reduction surgery diet used in one hospital is shown in Table 10, but there is no standard, widely accepted protocol for diet therapy post-bypass at present. Dietary restrictions must continue indefinitely after surgery to prevent weight regain, and patients will require lifelong use of appropriate vitamin and mineral supplementation to prevent deficiencies. Failure to adhere to a hypocaloric diet through such strategies as consuming large amounts of alcoholic or sugar sweetened beverages, melted ice cream, and many small but calorically dense meals will result in weight and fat gain.

While data suggests that bariatric surgery is more successful than non-surgical interventions, in promoting greater long-term weight loss and inducing initial remission of type-2 diabetes, there is a lack of evidence assessing the long-term risks, complications, and costs of bariatric surgery (238). It is important to note that post-surgery, patients are at risk for many nutritional deficiencies, which can negatively impact overall health, even in the context of weight loss. In one recent study that followed patients who had lost weight more than 50 lbs prior to body contouring procedures, those that had lost more than 100 lbs were more likely to suffer complications, with the effect being greater in those who had lost over 100 lbs from bariatric surgery compared to non-surgical means. Among patients who had bariatric surgery, the risk was highest for those who had had gastric bypass, and lowest in those who had has a lap-band or gastric sleeve (239).

Table 10. Post Gastric Bypass Surgery Diet Used in Tufts Medical Center (34)
Stage 1	One ounce of water per hour, typically in the hospital on the day of surgery
Stage 2	Non-caloric clear liquids, usually in the hospital the day after surgery (e.g., sugar-free Jell-O, flat diet soda, diet juice)
Stage 3	3-4 small meals per day, each consisting of a high-protein, no added sugar shake, such as Isopure or Sugar-Free Carnation® Instant Breakfast™ Water or non-caloric, non-carbonated clear liquids between meals Goals of this stage are to drink 64 oz fluid per day 50-60 grams of protein a day for women and 60-70 grams of protein per day for men This stage lasts 2-3 weeks
Stage 4	Small portions of moist, ground/pureed foods. Begin supplementing with a multivitamin plus minerals, Vitamin D with calcium (specifically calcium acetate), and sublingual Vitamin B ₁₂ Aim for 60-70 grams of protein per day This stage lasts 4-5 weeks
Stage 5	Small portions of low-fat (<3-5 grams per serving) or low-sugar (<14 grams per serving) solid foods At least 64 ounces of fluid per day Aim for 60-80 grams of protein Continue to take supplements Follow this 6-8 weeks after surgery and follow up with a Registered Dietitian

Note: Post Gastric Bypass Surgery Diet Used in Tufts Medical Center is adapted with permission from Melissa Page, MS, Rd, LDN, Weight and Wellness Center, Tufts Medical Center, Boston, MA.

There are many tools available to practitioners to aid in accessing obesity and its related health risks, as well as in determining appropriate treatment options. One such tool is the American Society of Bariatric Physicians Obesity Algorithm (292). Another useful tool is the algorithm in the “2013 Guideline for the Management of Overweight and Obesity in Adults,” which was created by the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and The Obesity Society (23).

5. Set Goals

Goal setting is an important part of achieving weight loss. This section outlines steps in goal setting that are successful for weight loss/ or weight maintenance.

5.1 Clarify Reasonable Goals

In 1998 the National Institutes of Health (NIH) issued guidelines recommending that healthcare professionals advise obese patients to lose weight. Evaluating the effect of these guidelines somewhat later, one study examined the proportion of obese patients and those that actually received advice between 1994 and 2000. The proportion of patients that were obese and received advice to lose weight from physicians during routine medical check-ups decreased from 42.3% in 1994 to 40.3% in 2000. Among those that did receive advice to lose weight had 2.8 odds of trying to lose the weight opposed to those who did not receive any advice (48). In addition to the NIH guidelines, in 2003, the U.S. Preventative Services Task Force (USPSTF) recommends that clinicians screen all adult patients for obesity and offer intensive multi-component behavioral intervention to those affected individuals. Primary care physicians play a critical role in screening adults for obesity and providing appropriate treatment (49). Even with these national recommendations and guidelines, it is still a challenge for healthcare providers to manage obesity (50). Another challenge is who is to pay for it, since many insurance plans do not, and many of those who are obese do not have the money to pay for treatment out-of-pocket. In 2009, less than 50% of obese patients received weight loss advice from physicians, citing many barriers to counseling and intensive treatment of obesity (50). This study concluded that there is a general lack of consensus about the best approach to weight loss; however, this can be improved if counseling skills are developed early during training of physicians in treating obesity (50).

As demonstrated in the study mentioned above, those patients who received advice to lose weight were almost three times more likely to attempt weight loss, so it is important for health professionals to discuss weight loss with their obese patients. The approach used by health professionals when treating obese patients and helping them to set goals is most effective when it is non-judgmental, respectful, and empathetic. This allows patients to feel comfortable with discussing their weight. The focus should be on an acceptable weight to achieve better health outcomes rather than simply reaching a lower body weight (51-53). Furthermore, health professionals must always keep in mind that overweight or obese patients might be hesitant to broach the topic of weight loss, but indeed want assistance in discussing, setting and achieving weight loss. By speaking and working with patients as partners in reaching their weight goal, health care providers can play a big hand in improving their patients’ health.

Understandably patients do not like the terms “obesity,” “fatness,” or “excess fat,” and are more receptive to terms such as, “weight” and “excess weight.” It may be difficult for health care providers to bring up the topic of weight loss, but for more tips and advice, healthcare providers can visit The National Institutes of Health’s Weight Control Information Network (WIN) at http://win.niddk.nih.gov/publications/talking.htm for more information on discussing obesity with patients and for some examples of how to bring up the topic of weight loss with patients.

Determining whether a patient is motivated to lose weight is pertinent and the first order of business, since patient involvement and investment are essential for weight loss success. If a patient is not ready to engage in weight loss, discuss the importance of weight maintenance and physical activity at visits until the patient is ready to begin weight loss therapy (24). Once the patient expresses the desire to lose weight, determine the patient’s weight-related goals and ascertain that goals are realistic and attainable. Agreeing on realistic goals facilitates maintenance of weight loss (54). Unrealistic goals should be discussed and made into more achievable ones. Additionally, patients need to be reassured that the counselor, or healthcare provider, is interested in their health as individuals. Providers should not assume that all of their obese patients’ health problems are weight-related (55), nor should they fail to treat these other problems even if the patients refuse treatment for the obesity. The health care professional’s job is to reduce health risks and improve quality of life to the greatest extent possible within patients’ wishes (56).

On their own, patients often choose to lose weight using drastic measures, such as fasting or very, very low calorie diets, to get fast results even though there is no evidence that they are the best for losing weight. Healthy weight loss is key for long-term weight maintenance (54;57). Intensive, very low-calorie diets (VLCDs; e.g., ≤800 calories per day and especially <500 calories) produce significantly greater initial weight loss, however, these results are often not maintained over time (58). In a study done in 2008, participants were enrolled in a program designed to help maintain weight loss. Participants all initially lost weight using one of three methods, VLCD, commercial programs or a self-guided approach. At the start of the study, those who had used a VLCD lost up to 24% of highest body weight in the last two year compared to those in the commercial programs and self-guided approaches who lost 17%. Results showed that those who used a VLCD regained significantly more weight than the other two groups by six months in the weight maintenance program. However, those who had lost weight using a self-guided method were able to maintain their initial weight loss with great success (58). It should be emphasized that the primary reason for losing weight is for better health outcomes; therefore, weight loss should occur by using healthy methods, ones that can be maintained throughout life. Concentrating on improving health outcomes and other risk factors rather than simply on weight loss is vital.

Once it is established the patient is motivated, health professionals should begin by working with the patient to set realistic, achievable, and sustainable weight loss goals (57). From the medical perspective, an ideal weight goal is one that will maximize heath related effects while minimizing disruption to the patient’s quality of life. This allows the patient to incorporate dietary changes into daily life and therefore incorporate them into their lifestyle to maintain weight loss. Physicians have access to measurements of weight-related risk factors that will be improved if weight is lost. Therefore they are uniquely qualified to define and communicate what a "healthier" weight should be for the patient. Nurse practitioners, registered dietitians, physician assistants, and others should reinforce the message the physician gives, and carry out the actual therapy.

There is no single target weight that will meet every one’s goals. Optimal weight reduction targets vary depending on the patient’s weight and co-morbidities. Progress toward healthier weight goals should involve a gradual approach that minimizes health risks and is timed to the patient’s level of readiness. Excess emphasis on aesthetic and cosmetic aspects of weight loss should be avoided. Most patients may have unrealistic ideas of how much better they will look with weight loss so it is important to stress the health advantages of even modest weight loss (i.e. a half-pound per week) (59). While some cosmetic improvement is possible with a weight loss of five to ten pounds, it rarely meets the patients’ expectations, which can be discouraging. Patients need to have a realistic weight loss target set for themselves, which they have developed during counseling session with a health professional. In time, greater weight loss may be possible if realistic goals are adopted, met, and sustained. The aesthetic and cosmetic effects of weight loss are "extra benefits." The primary medical concern is to help the patient lose enough weight to improve or maintain his/her health.

5.2 Adopt Realistic Goals that Include Health Objectives

A healthy target for loss is usually to achieve a weight loss of one-half to two pounds of body weight each week over six months (24 weeks), leading to a decrease of 5 to 10% in body weight from baseline. For example a 250 pound, 5’6’’ woman with a BMI of 40 that lost 12.5 pounds over six months, would have a 5% weight loss, and an ending BMI of 38.5. A 10% weight loss would result in a BMI of 36.3. The goal is to maintain this weight loss over time, and that is never easy. A weight loss of 5 to 10% is achievable and moderate enough to decrease some obesity-related risk factors, such as type 2 diabetes, hypertension, cardiovascular disease, and sleep apnea (9).

The recommendation of dieting for six months rather than a longer amount of time is a practical one because after about six months, most patients have great difficulty sustaining adherence to any diet, particularly if it is very rigorous. Weight plateaus as energy intake fluctuates and resting metabolic rate and energy output decrease. After six months of weight loss, patients should focus on maintenance of the weight loss through a combination of diet therapy, physical activity, and behavior modification. If successful, after several months they can start a weight loss cycle again. When patients do not engage in a weight management program that includes all three components, the risk they will regain all or some of the weight increases (57;60). Most individuals regain one-third of their lost weight in one year, and nearly half return to their original weight within five years (54). The more frequently a patient has contact with his/her health care provider, the weight loss and maintenance outcomes tend to be more successful (57).

5.3 Define Successful Outcomes for Weight Reduction with the Patient

Patient weight goals depend on their motivation and their perception of health risks, in addition to other aspects of their lives unrelated to health outcomes. Some patients simply are not motivated to lose weight, or they may be motivated but unwilling or unable to make any changes at present. It is unreasonable provide an unmotivated patient a weight loss goal that they do not wish to achieve. Instead it would be beneficial to come to an agreement about the steps the patient is willing to take to begin to improve their health. One of the newer techniques used in counseling is “motivational interviewing” (MI); an egalitarian, empathetic approach to counseling. It uses specifics strategies, such as reflective listening, positive affirmation, and agenda setting to engage the patient in health behavior changes that will facilitate weight loss. The goal in MI is to assist individuals to work through their ambivalence about behavior change and to find what motivates them internally. Patients understandably often have strong aversions to the weight loss process, so MI counselors reflect the patient’s doubts and work through those with the patient. The process also provides opportunities for the patient to voice concerns about remaining overweight or gaining even more weight, which might serve as motivation to begin losing weight or gaining more control of their weight. MI can be more effective than the counselor simply stating facts to counter the beliefs and doubts of the patient and often enables patients to develop their own reasons and plans for change. Individuals are more likely to accept and act upon their own choices and opinions when they voice them themselves (30).

For patients who are already motivated, the weight loss process can begin much faster because the patient is already willing to make changes. However, it is important to check what they think they will accomplish and when. Their patients’ weight loss targets are often unrealistically low and their time frames unrealistically short (e.g., targets of 25% or more of body weight in a few weeks rather than many months). Numerous studies have shown that obese individuals hope to lose 25 to 35% of their initial weight within a year or less after beginning obesity treatment. Realistically, patients lose only 5 to 15% of their initial weight over a year after beginning any kind of obesity treatment. Unfortunately, many dieters still maintain unrealistic standards even when they are repeatedly informed that their goals may be unrealistic (61).

For example, in one recent assessment, the before-treatment weight loss goals of 45 obese women were assessed, and the women were randomized into a behaviorally based weight-loss program over 48 weeks. While 8 to 10% of weight loss would have been a success from a medical perspective, the women identified a loss of 32% of their body weight as ideal. At the conclusion of their 48-week treatment, the women lost an average of 16% of their total body weight. Even though their weight loss was more than medically expected, the women collectively considered this loss to be “disappointing” (54). This study illustrated how most patients’ ideal weight loss goals are unrealistic and often two to three times what most patients achieve (62). For these types of patients, counseling on more realistic targets and time frames is helpful.

Because patient weight goals are often very different from those of their healthcare providers, health professionals must clearly understand patient expectations for treatment and understand the rationale behind these patient expectations. It may be necessary to work with the patient to re-evaluate changes in expectations over the course of treatment (54). Providing patients with verbal and written information on how much weight they can expect to lose with obesity treatment is helpful in communicating and setting realistic weight loss goals (61). When patients’ ideals of drastic weight loss are not met within their preferred time frame it leads to disappointment and frustration, so these dramatic goals must be addressed at the onset of treatment. It is also important to praise patients once they begin to make positive behavior changes. It helps them feel as though their efforts have been acknowledged and motivates them to maintain their new habits and continue to lose weight or stop gaining more weight. Health professionals should be sure to frequently remind their patients of the health benefits that a 5 to 10% weight loss will offer.

5.4 Define Dieting Success in Broader Terms than Weight Loss Alone

Definitions of success are always patient-specific, but health professionals should emphasize the importance of health outcomes rather than how the patient would like to look as their definition of weight loss success. The definition of successful obesity treatment includes goals other than weight loss, and these broader health goals need to be communicated to patients. The reduction of risk factors and co-morbidities, even if weight is not lost, is a "success" from the health standpoint. For some patients, prevention of further weight gain after years of a slow, steady increase in weight is “progress.” The maintenance of a reduced weight, even if it is still within the range of obesity as clinically defined, is also a "success". Some outcomes to focus on include improved metabolic profiles such as, lower blood pressure, serum cholesterol, or fasting blood glucose. The following health behavior changes also denote success: increased daily physical activity and fitness; greater healthfulness of eating patterns, such as more consumption of fruits, vegetables and fiber; and reduction in dietary fat. Changes in specific unhealthful habits such as smoking, or overindulgence in alcoholic beverages, are also reasonable measures of success that may help enhance self-esteem, self-efficacy, quality of life and functional capacity (24;5).

5.5 Set an Individualized "Healthier Weight" Target with the Patient

Patients can be unreasonably hard on themselves and fear that losing weight requires drastic measures. An initial healthy weight goal of 1-2 BMI units often requires much less extreme measures than patients think. For example, a 5’4’’ woman weighing 250 pounds with a BMI of 43 losing 5% of her body weight, or 12.5 pounds, will have an ending BMI of 41. This amount of weight loss could take up to 25 weeks, if she loses one-half a pound per week. Weight loss of one-half a pound to two pounds per week is reasonable and offers the best chance for long-term success, but for extremely heavy people, this may take many months or years. However, as mentioned before, weight loss of even 10% of initial body weight, if sustained, significantly reduces risks of coronary heart disease and other co-morbidities (9;63). Obese patients often expect to lose 25% to 35% of their initial weight over the first year of obesity treatment. Dieters often maintain these expectations even when they are repeatedly informed that their goals are likely unrealistic —even with pharmacological treatment, so the message needs to be repeated (61).

5.6 A Reasonable Target: 10% Loss of Body Weight over 6 Months

A 10% weight loss target can be achieved in most patients with a caloric deficit of 500 to 1,000 calories per day, leading to losses of one pound to two pounds per week. For women, a weight reduction plan of eating approximately 1,000 to 1,200 calories per day is suitable. According to the National Institutes of Health and the National Heart, Lung, and Blood Institute, a 1,200 - 1,600 calorie allowance for men or women who weigh 165 pounds, or more, and who exercise regularly is recommended. These calorie amounts along with increased physical activity and behavioral modification will likely produce a caloric deficit to achieve the targeted weight loss plan of one to two pounds per week (64).With a caloric deficit of 500 to 1000 calories per day, if followed with perfect adherence, after six months, weight loss of 26 to 52 pounds would be expected. However, in reality, losses are usually between 20 to 25 pounds, since adherence is never perfect (65).

A decrease of one BMI unit usually represents a loss of 10 to 15 pounds, but the exact amount depends on height and weight. A decrease in two BMI units over six months is another way of stating a weight loss goal. Reductions of this magnitude in weight usually decrease several weight-related risk factors such as blood glucose and blood pressure, which should result in better overall health. In addition, patients’ clothing is likely to fit better and their appearance should be trimmer. If further weight reduction is necessary after 10% of initial body weight is lost, it can be attempted with an increased calorie deficit after prior weight loss has been maintained for several months. Medical nutrition therapy for obesity should last at least six months or until weight loss goals are achieved. After that it is vital to begin a weight maintenance program that includes the same three components used for initial weight loss, diet, physical activity, and behavior change to help prevent weight regain and maintain the patient’s new, healthy lifestyle (57). In addition, the patient should have a strong social support network of encouraging friends and family, and/ or participate in a group where others are also undergoing weight loss treatment. With a strong social support network it is easier for patients to continue his/her healthier lifestyle (66).

5.7 Set an Increased Physical Activity Goal

Physical activity is important in weight loss. Physical activity and exercise are not synonymous. Both are desirable but the first is essential. If left on their own, most dieters become more sedentary during weight loss, especially if diets are very low in calories. This is because a markedly negative energy balance reduces exercise tolerance, the body’s maximal power output and increases the body’s sense of perceived exertion (67). Therefore, conscious efforts to increase physical activity while dieting should be attempted. However, physical activity alone, without a reduction of calories, only induces modest reductions in body weight. Few studies to date have incorporated enough physical activity to achieve even a 5% weight loss using a physical activity intervention alone. When physical activity is paired with energy restriction, it has a synergistic effect on weight loss. Despite its modest effects on weight loss, physical activity is also essential for improving health-related outcomes relevant to many obesity related co-morbidities (e.g., heart disease, type 2 diabetes, and possibly some cancers)(68). Physical activity is also vital in preventing weight regain and may enhance quality of life (69). There is a strong association between physical activity at follow-up and maintenance of weight loss. Data from the National Weight Control Registry, a registry of more than 3,000 individuals who have successfully maintained at least a 30-pound weight loss for a minimum of one year, shows that 90% of the individuals report that physical activity is crucial to their long-term weight maintenance. They report expending, on average, 2,700 calories per week in exercise, the energy equivalent of walking four miles seven days a week (70).

5.8 Individualize the Diet and Treatment Program

Evidence-based reviews of successful weight control techniques increasingly emphasize the importance of individualized, multidisciplinary care in addition to realistic goals that are focused on health-outcomes and making permanent lifestyle changes, including an increase in physical activity (65;71).

The specific factors that induce a chronically positive energy balance differ among individuals. Daily lifestyle, environment, resources, and social situations may vary considerably. Weight loss strategies must, therefore, be individualized in order to promote adherence and success (5). No single diet works for everyone. Different dietary approaches for maximizing adherence are successful to varying degrees in different individuals. If asked, patients can usually identify some strategies that have worked for them in the past and health professionals can build a program starting with these strategies as starting points. Previous pitfalls can also be identified and the new weight loss strategy can be tailored to avoid them. Candidates for weight reduction should discuss the approach that best suits their needs with their physician, dietitian, or other health professional. In addition to energy content, individual food selections, meal frequency and many other factors can be tailored to make the diet better suited for the individual. Some factors to consider include the diet’s cost, convenience, how it approaches treatment of co-existing health conditions, and whether it assists patients in adopting strategies for healthful life-long weight maintenance (72;73).

Many overweight patients have already tried many times to lose weight on their own. For example, in the United States 50 – 70% of US adults are trying to lose weight (74). Self-directed efforts are usually motivated by aesthetic or social rather than health-related reasons. The goals they adopt are often unrealistically ambitious, the information they obtain on weight management is often inaccurate, and the motivation and support they receive is often inadequate. Solo efforts often fail and lead to discouragement and a sense of futility (75). The vital role of health professionals is to provide motivation, information, counseling, and support for patients to be successful.

Throughout weight loss patients must be counseled on sound eating patterns. Some dietary education topics that should be discussed to help them are listed in Table 11. The National Institutes of Health (www.nutrition.gov), the American Dietetic Association (www.eatright.org) and other organizations, provide materials, checklists, guidelines, menus, and recipes to assist in such patient education (4;48). Resources for health professionals and for patients can also be accessed at websites such as myplate.gov, the American Heart Association (http://www.americanheart.org/), American Diabetes Association (http://www.diabetes.org/), the American Cancer Society (http://www.cancer.org/), and the American Dietetic Association (http://www.eatright.org/).

Table 11. Checklist of Nutrition Education Topics to Cover in
Counseling Patients on Weight Management (15:57)

Dietary Interventions

Energy values of different foods
Food Composition (calories, fats, carbohydrate, fiber, protein)
The Dietary Guidelines for Americans
Portion control and standard serving size (individualized)
Meal planning and food preparation
Recipe modification
Avoiding over consumption of foods with high energy content but little nutritional value
Hydration status and limiting alcohol consumption
Discuss health risks of obesity and rapid weight loss (monitor health with a team of healthcare providers)

Physical Activity

Increases in physical activity (individualized)

Behavior Change

Reading nutrition labels
Cooking more meals at home
New habits of food purchasing
Mealtime strategies to avoid overeating
Eating strategies for restaurants and social situations
Awareness of physiological hunger and satiety cues
Awareness of eating and emotions
Establishing achievable short-term and medium-term goals
Tracking intake and physical activity to keep accountability and records of progress
Seeking support

Patients should maintain daily records of their food and beverage intake; this helps to cue them to restrain themselves, and also the record is a visual reminder that it is important to watch one’s intake. Some may also wish to include their mood at the time they ate in order to help in recognizing reasons for eating beyond hunger. Record keeping often increases awareness of consumption, and promotes dietary adherence. Patients should be encouraged to review food records each week and to identify any patterns related to eating and behavior that they can work on for the next week.

6. Plan the Weight Reduction (Energy Deficit Phase) of Weight Control

This section will cover guidelines for the calorie-deficit phase of weight loss.

6.1 General Principles

For an individual already overweight, successful weight control first requires a hypo-caloric phase during which dietary intake is decreased while energy output is increased (or at least not decreased). This phase is referred to as the ”weight loss” "energy deficit" or "hypo-caloric phase" of weight loss.

The essential components of weight loss, regardless of type of diet, are decreased energy intake, increased energy output through physical activity, behavioral modification and alterations in the environment that foster all of these measures. Although this chapter focuses on dietary measures in the treatment of obesity, all reasonable weight control programs should also include physical activity and behavioral modification.

6.2 Size of Caloric Deficit Needed to Lose Weight

Obesity results from the accumulation of excessive body fat, which is stored as adipose tissue. An energy deficit of approximately 3,500 calories is required to lose one pound of fat. However, there are several factors that can influence this particular number. These include compensatory changes in resting metabolism, the energy cost of work, and discretionary physical activity, which can sometimes alter this figure by 100 to 200 calories. Over the long-term, this relationship of 3,500 calories per pound of fat holds up quite well. Thus, it is the size of the energy deficit between basal energy needs and the energy output that determines the slope of decline in adipose tissue over time. How well this energy deficit is maintained throughout the weight loss period is dependent on a multitude of factors (76). In addition, creating a calorie deficit using this rule of 3,500 calories per pound may not be applicable to everyone. Work by Dr. Kevin Hall has shown that initial body fat as well as the magnitude of weight loss can influence the applicability of this rule (76).

As previously mentioned, a reduction of 500 to 1,000 calories per day is recommended to achieve a weight loss of approximately one to two pounds of body weight per week (i.e. -3,500 to -7,000 calories total). Cutting down on alcohol, dietary fats and/or sugary caloric carbohydrates is a practical way to produce this deficit (70).

6.3 Goal of the Energy Deficit Phase

The goal of dietary treatment of obesity during the weight loss (energy deficit) phase is to decrease body fat stores without unduly depleting lean body mass or otherwise compromising health. Lean body mass includes skeletal muscle and vital organs. During weight loss, some lean muscle tissue is always lost in combination with the fat loss, but the goal is to keep this loss to a minimum (64). While weight is shed, body stores of other nutrients such as water, vitamins, minerals and electrolytes must be maintained. Fortunately, dietary strategies are available to minimize loss of lean tissue and other nutrients.

A systematic review found that increased lean tissue is lost if the energy deficit of the diet is too large in combination with rapid weight loss. In contrast, inclusion of exercise (both cardiovascular and resistance) and adequate dietary protein (60 grams per day, ranging from 0.8 g - 1.5 g per kg of body weight) helps to minimize lean tissue loss (73). These dietary strategies should be incorporated into dietary treatment plans to minimize lean body mass reduction and maximize fat loss.

7. Troubleshoot Diet Failures

There are many factors that contribute to patients losing less weight than expected. This section describes how health professionals can work with these patients and address the issues that are coming into play.

7.1 Keep Food Records: Food Intake Varies from Day to Day and it is Easy to Forget to Diet Every Day

People vary in their eating patterns from day to day. Weight reduction prescriptions are such that the patient should aim for a caloric deficit of approximately 500 calories per day, which would achieve weight loss of about one pound per week. This can seem like somewhat of an abstract since the patient may not know how many calories he/she is eating in the first place. Since most people vary in their food intake from day to day, they have difficulty recognizing if they are eating less than they were previously. For this reason, simply urging patients to "eat less" of certain foods in general, is unlikely to produce clinically significant weight loss. Specific advice is more appropriate and easier for the patient to achieve. Examples of specific actions that decrease caloric intake include, cutting down portion sizes of high calorie, frequently consumed foods; avoiding appetizers; eliminating a second cocktail; replacing a second serving of steak at dinner with vegetables; or ordering roasted, baked, grilled, or steamed foods instead of fried, deep fried, sautéed, or creamed items when dining out.

Patients should also be advised to increase their intake of foods that are low in totally calories while also increasing the fiber content of their foods and replacing high-fat food with minimally processed, carbohydrates or proteins. A diet rich in food that is low in energy density, such as fruits, vegetables, and soups, will reduce caloric intake while also promoting satiety. This strategy is thought to be superior to a fat and portion restricted diet. A 2010 study evaluated the energy density of daily intake for three different groups. One group comprised overweight adults, another was of normal weight adults and the third consisted of weight loss maintainers (who had lost  10% of maximum body weight and kept it off for  5 years). Dietary intake was collected via three 24-hour phone dietary recalls and energy density was calculated using three different methods. Results showed that those in the weight loss maintainer group consumed significantly less energy per day (49). When the energy density of food is decreased, but the volume of food remains the same, calories consumed will decrease. In one study, the energy density of foods was lowered by 30%, consequently, daily energy intake also decreased by 30% (77).

Providing calorie recommendations, and instructions to keep food and physical activity records, will help patients see what factors influence their weight. Patients who record their daily food intake (i.e., food item, portion, calories, time of eating, and fat grams, if desired) as well as their physical activity for the day, are more successful in weight loss and weight maintenance efforts than those who do not (78). Some patients find it helpful to write down their emotions during their meal times to help assess whether the patient is eating out of emotion or out of hunger. The National Weight Control Registry data indicates that frequent self-monitoring of caloric intake and weight helps patients to maintain their new lower weight (79).

Patients who are able to self-monitor are more successful in weight loss efforts than those who do not self-monitor. Self-monitoring fosters awareness, an essential initial step in behavioral change. The Handbook of Assessment Methods for Eating Behaviors and Weight-Related Problems states that, “It is well established that self-monitoring or recording daily intakes via food records is a useful tool in weight loss programs” (80). Furthermore, the interplay among awareness, self-observation, recording, and self-evaluation can enhance self-management by improving how individuals attend to their health. A common denominator among all successful weight losers is self-monitoring (57;78;79;81). Patients who use food records report they have a heighted awareness of their eating behaviors, they recognize the need to make significant dietary changes and they are more able to “stay on track.” In addition, their label reading, fat and calorie counting, and portion determination skills are improved (81). These are all important skills that overweight or obese individuals need to lose weight and/or maintain their weight at lower levels. These are also skills for making healthier lifestyle choices throughout the rest of their lives, thus improving their overall health.

7.2 Self-reports of Energy Intake are Almost Always Underestimated

The average, healthy, adult, American male consumes approximately 2,800 calories per day, and the average female about 1,800 calories. Yet, such intakes are seldom accurately reported in a diet recall or food log. Usually, the recall or food log will show a much lower calorie intake. Reporting energy intakes is difficult, even for individuals who have been trained to do so accurately. In other words, people are widely unaware of what or how much they are eating on a daily basis. Even small omissions or inaccurate portion size reports could subtract hundreds of calories from the total calorie consumption of the day. Several days of observation are necessary to achieve accurate calorie intake since energy balance is achieved over weeks, not days. Thus, a report from any given day is certain to have a considerable amount of random as well as probably systematic error when used to estimate usual calorie intake.

Underreporting of energy intakes is common and usually off by a large margin (20%) in virtually every patient, and particularly so among the overweight population (82). Objective biomarkers of energy output such as doubly labeled water indicate that underreporting could be as great as 1,200 calories per day in very obese persons (83;84). Subjective reports of energy intake are often so low that if they were actually true, those patients should be losing weight. But in fact, they are gaining weight. It is biologically impossible to gain weight on a hypo-caloric diet, so underreporting must be considered (85).

The most common problem arising from these errors is that patients’ actual weight loss is usually less than was desired. This could be a result of underreporting and underestimating intake. Typically, when overweight people report their intakes by recall, they often underestimate their intakes by 30 to 40%. These patients are likely to make similar mistakes in underestimating their intakes on reducing diets because of difficulties in portion size judgment, forgetting, the social desirability of reporting adherence to the prescribed regimen, and other factors. This can be explained by the “flat-slope phenomenon,” which describes how individuals with a high intake of food tend to underreport their intake while those with a low intake of food tend to over report (48). For example, many people underestimate or forget that their very large food intake on weekends “counts”; or they forget to count alcohol, snacks, or something they may have had a second helping of. Thus, on a 1,200-calorie diet, actual consumption may be 2,600 calories or more on some days, and weight loss understandably slows.

Methods for assisting dieters in minimizing diet recall errors would be to use household measures or weighing scales to determine the portion size consumed more accurately, in addition to the use of food diaries. Portion-controlled liquid meal replacements, frozen low calorie entrees, and other foods that are fixed in portion sizes might also be helpful in not only portion control but also in reporting that intake because the portion size is usually labeled (see 10.3 Formulas and Meal Replacements).

Consistency in reporting intake does not necessarily mean that the reported intake is accurate, which is especially true for those who are morbidly obese. Underreporting is also especially pronounced among women, smokers, and those of low educational and socioeconomic status (83). In addition, those who underreport tend to be consistent under-reporters, and so they are difficult to detect since the records are all similar (83). In spite of these limitations, patients who keep food journals or diaries, are more successful in losing and/or maintaining weight loss than those who do not (78). Additionally, self-reports are useful for the patient and counselor alike to obtaining clues on dietary patterns that may be helpful in working through barriers to weight loss.

7.3 Remind Patients to Stay Active: Self-reports of Energy Output Tend to be Overestimated

Self-reports of energy output as measured by physical activity questionnaires have been validated using doubly labeled water methods. Some lengthy questionnaires used for research purposes are quite accurate at estimating physical activity (86). However, the shorter questionnaires, used clinically, are not accurate for individuals (87). As is the case with dietary recalls, physical activity questionnaires may be useful for self-monitoring, but should not be used for prescribing or assessing energy intakes or outputs exactly. Motion sensors (pedometers or accelerometers) have become popular in recent years. Accelerometers and pedometers provide objective physical activity measurements and are sensitive to walking (88). Either is a worthwhile purchase to help in self-monitoring of physical activity.

Accelerometers measure the body’s acceleration in one direction for long periods. In contrast to the pedometer, an accelerometer distinguishes between different walking speeds and intensities. Many accelerometers also record the amount of steps taken, allowing comparison with pedometers. Accelerometers have been validated as accurate forms of measurement in regards to steps counted (88). On the other hand, pedometers are less expensive because they measure only step count and not walking speed so this may be a better option for someone who is not willing or able to purchase an accelerometer. Unfortunately, pedometers are less sensitive to detecting steps if a person is walking slowly (e.g., less than two miles per hour). When walking at this slow of a pace, pedometers underestimate step count by approximately 50 to 90%. However, if a person is walking above 3.5 miles per hour, most pedometers approach 100% accuracy in step count (89). If a patient chooses to purchases a pedometer, piezoelectric pedometers are best because they are more accurate than spring-levered pedometers. This is especially true for obese or elderly individuals, who are more likely to walk at a slower pace, because these pedometers measure slow walking speeds more accurately (89;90).

If a motion sensor, either a pedometer or accelerometer, has a calorie counter built in, it could be inaccurate. Rather than focusing on the calories burned according to the motion sensor, patients should focus on the number of steps walked per day. Goals of a certain number of steps each day can be prescribed and patients are able to monitor their progress on their own. One common step count goal is 10,000 steps per day. However, most patients need to work up to that number so they should set smaller, more achievable goals until they feel they can reach the ultimate goal of 10,000 steps. The basic point is that if the patient can be induced to walk 10,000 steps a day or to gradually increase the number of steps he walks no matter how low it is, progress is being made, and the finer points of absolute accuracy can be disregarded. The ability for patients to quantify physical activity in a tangible way fosters commitment, encourages performance, provides a realistic goal, and eventually may provide a feeling of self-accomplishment.

7.3.1 Compensatory Decreases in Energy Output Occur on Most Reducing Diets

Unconscious compensatory decreases in physical activity usually occur on reducing diets, particularly if diets are at a very restricted calorie level. These decreases in physical activity result in slower weight loss. As a rule of thumb, if a person is decreasing calorie intake by 500 calories, there is a corresponding decrease in energy output of about 165 calories. This is a result of decreased resting metabolic rate, decreased discretionary physical activity and a decreased in energy used moving the body, which results in only at 335-calorie deficit rather than a 500-calorie deficit. So, the caloric deficit may be less than anticipated and actual weight loss becomes less than desired (91). Energy balance seems to be more strongly preserved during energy deprivation than during energy surplus, which impedes weight loss to a greater extent (92-94). This retarding effect on weight loss may be due to compensatory slowing down of resting basal metabolic rate, decreased non-obligatory physical activity and decreased thermogenesis. By including physical activity during the weight loss phase, these alterations can be alleviated to some extent, through greater energy output and preservation of lean tissue (10). Health professionals can use this information as an additional incentive for patients to maintain physical activity.

7.3.2 Physical Activity Guidelines for Americans who are Overweight

All weight loss programs should include physical activity. According to the 2008 Physical Activity Guidelines for Americans, recommendations for weight loss include engaging in 45 to 75 minutes of moderate-intensity activity per day. This can include activities such as, walking at least three miles per hour, participating in water aerobics, ballroom dancing, or gardening. Alternatively, individuals could instead participate in 22 minutes of vigorous activity per day, such as swimming, jogging, jumping rope, or hiking. Once an individual loses weight, physical activity and exercise are still important for maintaining weight loss. For weight maintenance, 60 minutes of moderate activity per day or 30 minutes of vigorous activity per day is recommended. In addition, weight resistance activities, which involve all the major muscle groups, are recommended for two or more days per week. If these recommendations cannot be achieved, it is helpful to stress to the patient that t some activity is better than none. These national recommendations should be used as goals but it may take time to reach those goals. It is a good idea to start where the patient feels comfortable and confident that they can accomplish their goals before overwhelming them with requirements of great amounts of exercise.

Dramatic decreases in weight often occur on reducing diets, particularly in the first few weeks. This is especially true for those on severely hypo-caloric regimes (with deficits of 1,000 calories per day), those on ketogenic diets, and those on very low carbohydrate diets (95;96). The type of fluid shift (loss or increase of fluid) depends on the caloric level and macronutrient composition of each diet. For example, high protein, low carbohydrate diets increase obligatory urine volume due to greater urinary loads of nitrogen, ketones, and other solutes (particularly sodium, if they are low in sodium), and will result in increased fluid losses . Increased loss of lean tissue is also associated with large fluid losses because of loss of nitrogen from tissues, such as muscle, along with water, which comprises most of the lean tissue. Fluid losses are also more apparent on hypocaloric regimes that are very low in carbohydrate (<100 grams/day and especially <50 grams of carbohydrate), since they are insufficient to replete glycogen stores, and glycogen consists largely of carbohydrate and water. Shifts in water balance may cause very dramatic deviations from the usual linear slope of weight loss. They may also cause rapid weight accumulation over just a few days if there is a period of non-adherence. This is a result of the body storing glycogen and water when the dieter eats a large carbohydrate load after a period of carbohydrate deprivation. Fluid accumulates rapidly because for every gram of glycogen that gets stored, three grams of water are stored with it. Thus, gains or losses of glycogen are associated with large changes in body water balance and water weight. These shifts can be sudden and alarming to patients (96).

7.4.1 Weight Loss Varies with Water Balance Shifts

Water balance and weight often shift during the initial period of weight loss programs, particularly on very low carbohydrate diets. As glycogen stores are depleted in response to reduced carbohydrate intake, there is an increase in fluid loss, which produces an initial and often dramatic weight loss. However, the steep rate of initial weight loss will not continue. Patients should be reminded that one-half to one pound per week of fat loss is a realistic, achievable goal that will improve their health. Additionally, they should be told that it is difficult to continue losing weight at such a rapid pace. Since glycogen stores are likely depleted, and the initial diuresis has already been achieved, there is less water weight to lose. A recent position paper on Weight Management concluded,

7.4.2 Remind Patients that Fat Loss and Weight Loss Do Not Always Track over the Short Run, Although They Do Over the Long Run

In the long term, fat loss and weight loss closely parallel each other. However, this is not necessarily true at the beginning of obesity treatment. The amount of weight that is lost over time, particularly in the beginning (the first several days) depends not only on the energy deficit from metabolic needs, but also on adherence to the weight reduction plan (76). Additionally, shifts in water balance may be considerable over the short run. These shifts can make it seem as though more fat is being lost than in reality. It is important to educate patients about these patterns so they are aware of what is happening to their body as they go through their weight loss program.

8. Set the Caloric Level of the Reducing Diet

If a caloric level of a weight-reducing diet is to be set, the two general approaches include calculated-deficit and fixed-calorie diets. With calculated deficit diets, current daily caloric needs of the patient are either measured or estimated, and the deficit is derived by subtracting from those needs (70). Fixed-calorie diets have a pre-determined recommendation for total daily caloric intake, based on caloric levels that produce weight loss in clinical trials (70) Fixed-calorie diets include low-calorie diets (LCD) and very-low-calorie diets (VLCD). Calculated-deficit and fixed-calorie approaches are discussed in this section. Another approach, in which an ad libitum diet designed to produce a caloric deficit through restriction or elimination of particular foods, is described in section 9.

8.1 Calculated-Deficit Diets

From the clinical standpoint, hypocaloric diets must be defined in terms of the energy needs of the individual and the deficit that will be created, since it is the size of the energy deficit that will determine the physiological effects expected. Ideally, energy needs should be based on resting metabolic rate (RMR), while taking into account level of physical activity. If possible, RMR should be measured (e.g., with indirect calorimetry) (70), since it can vary significantly from prediction calculations in obese individuals (97). Products such as the MedGem^TM indirect calorimeter can be used in an office environment to quickly, easily, and accurately estimate a patient’s RMR (98), although caution should be used, since it may over-estimate RMR in overweight individuals [Anderson et al., 2014]. However, “if RMR cannot be measured, then the Mifflin-St. Jeor equation using actual weight is the most accurate for estimating RMR for overweight and obese individuals” (57;70). According to the Academy of Nutrition and Dietetics Evidence Analysis Library (EAL), the Mifflin-St. Jeor equation accurately predicted RMR using actual body weight within +/- 10% of measured RMR in 70% of obese individuals (57). Of the remaining 30%, 9% were overestimations and 21% were underestimations. Table 12 presents this equation. The individual error range was a maximum overestimate of 15% to a maximum underestimate of 20%” (24). While the Harris-Benedict and WHO equations are often used in clinical practice with reasonable accuracy, results have been mixed regarding their applications to individuals who are overweight or obese.

After calculating the patient’s RMR, his/her RMR should be multiplied by an appropriate physical activity factor to provide a baseline daily caloric level for weight maintenance. Once a baseline caloric level is configured, the patient’s recommended calorie intake should be reduced to facilitate weight loss. Reducing the calorie level by 500 calories is a common strategy to yield a weight loss of approximately one pound per week, although reductions of up to 750 calories per day are sometimes used (31). Another approach is to reduce current caloric intake by 30% (31). However, depending on the patient’s BMI and current intake, a larger reduction in calories may be needed, as described in the following sections. Calculations for estimating energy needs and various physical activity factors are provided in Table 12.

Table 12. Estimating Resting Metabolic Rate Using the Mifflin-St. Jeor Equation (15;57;70)
Males >19 years old RMR = (9.99 X actual weight) + (6.25 X height) – (4.92 X age) + 5*use weight in kilograms ( kilogram), height in centimeters (cm).
Females>19 years old RMR = (9.99 X actual weight)+ (6.25 X height) – (4.92 X age) – 161*use weight in kilograms (kilogram), height in centimeters (cm).
Activity Factors for Different Physical Activity Levels
	SedentaryLight physical activity associated with typical day-to-day life.	Low ActiveWalking about 1.5 to 3 miles per day at 3 to 4 miles per hour, in addition to the light physical activity associated with typical day-to-day life.	ActiveWalking more than 3 miles per day at 3 to 4 miles per hour, in addition to light physical activity associated with typical day-to-day life: 60 minutes of at least moderate intensity physical activity	Very ActiveWalking more than 7.5 miles per day at 3 to 4 miles per hour, in addition to light physical activity associated with typical day-to-day life: 60 minutes of at least moderate to vigorous intensity physical activity
Males	1.00	1.11	1.25	1.48
Females	1.00	1.12	1.27	1.45

The major determinant of weight loss on reducing diets is size of the actual, and not the prescribed, caloric deficit. Thus, if energy needs can be measured or estimated with reasonable accuracy, then a calculated caloric deficit would be the preferred method (70). Once caloric needs for current weight maintenance are determined, the deficit can be calculated by subtracting 500-750 calories, or with a 30% caloric reduction. Diets that reduce caloric intake relative to energy expenditure result in weight loss, regardless of macronutrient composition (31).

Recent advances in methodology such as mathematical modeling have demonstrated the amount of weight loss may not be as high as one might predict based on energy balance alone (99). This is due largely to metabolic slowing that occurs with weight loss, even after accounting for declines in lean tissue (100). As weight is lost, total energy expenditure declines; so fewer calories are needed to maintain weight (101). Thus, ideally, RMR should be measured periodically as weight is lost, and caloric intake recommendations adjusted accordingly. Physical activity may ameliorate some of this decline, but not all (100).

8.2 Low-Calorie Diets (LCDs): 1,000 to 1,200 Calories for Females, 1,200 to 1,600 Calories for Males

The caloric level of the diet requires attention first and foremost; after this, other characteristics of the diet can be considered. Diets that reduce caloric intake to about 1,200 to 1,500 calories in women, and 1,500 to 1,800 calories in men will result in weight loss if they are adhered to perfectly, regardless of their macronutrient composition (102; 31). This is because such caloric levels will result in a caloric deficit for most overweight and obese adults. The National Institutes of Health have recommended low calorie diets of 1,000 to 1200 calories for women, and 1,200 to 1,600 calories per day for men, although adherence may be difficult with lower caloric intake (24;65). Either way, the rationale is that on such regimens, a deficit of approximately 500 to 1,000 calories per day will be created, which should result in a slow progressive weight loss of one to two pounds per week. Two sample menus and other materials at 1,200 and 1,600 calories and many aids to assist the physician are provided in the NIH monograph (65). The ChooseMyPlate.gov website also provides such materials and worksheets that can be accessed by consumers, and guide them daily through their weight loss.

It is important to recognize that when using fixed calorie reducing diet plans, that even with perfect adherence, individuals will vary greatly in their weight loss. This is because their resting energy needs and physical activity, and thus energy outputs, often differ markedly, and may fluctuate even within an individual.

The National Institute of Health’s Obesity Initiative sponsored an evidence based review of low calorie diets (65). It found that on average, diets such as these reduced body weight by an average of 8% over three to 12 months of treatment, and that the losses were accompanied by decreases in abdominal fat, which is the type of adipose tissue deposition that is associated with highest chronic disease risk. However, no improvements were noted in cardio-respiratory fitness as measured by VO2 max unless the dieters also increased their physical activity (24).

There are many pre-packaged meals on the market that fit into the low-calorie diet category, including Healthy Choice®, Lean Cuisine®, and Smart Ones®. Some patients find them useful for one or more meals a day since they provide a measured and moderate amount of calories per meal. Weight Watchers®^TM, Jenny Craig®^TM, and NutriSystem^TM are more structured commercial programs that also can provide pre-packed, pre-portioned food options. See Tables 21 and 23 for a list of these programs and products.

8.3 Very-Low-Calorie Diets (VLCDs): ≤ 800 Calories

VLCDs supply 800 calories or less, a total of 50 to 80 grams of protein, 100% of the Reference Daily Intake (RDI) for vitamins and minerals per day, and are designed to produce very rapid weight loss while still preserving lean body mass (39). VLCDs are prescribed as a form of intensive diet therapy, which require close medical supervision, and should not be used long-term. They are intended to induce quick and significant weight loss of about 3-5 pounds weekly, or 14 to 21 kilograms over a short time (11-14 weeks). VLCD’s are sometimes used to provide a jump-start to further obesity treatment. This is typically done through meal-replacement liquid diets (6), as described below. Any diet, regardless of its caloric level, that provides less than half of an individual’s energy needs can be considered a VLCD for that individual. However, virtually all adults have energy needs that exceed 1,000 calories per day, and therefore any diet below 500 calories, and for most individuals, diets below 800 calories, are VLCDs. Depending on a person’s caloric requirements, other regimens that are higher in calories may also be VLCD for some people with very high energy needs using this same rule of thumb; for example, a 1,200 calorie diet prescribed to a man whose usual intake is 3,000 calories would also qualify as a VLCD..

8.3.1 Uses and Candidates for Therapy

These VLCDs are reserved for special uses and for individuals at high risk because of their potential for greater adverse metabolic effects and the consequent need for more extensive medical monitoring. Possible side effects range from fatigue, constipation, nausea, or diarrhea to more serious risks such as ketoacidosis and gallstones (section 8.3.4). VLCDs are often used when the health risks from obesity are particularly acute and threatening so that it is imperative to lose weight. Other individuals can usually reduce just as well on a LCD with less risk and discomfort (103). According to the National Task Force on the Prevention and Treatment of Obesity, VLCDs in patients with BMIs >30 are usually effective in promoting significant short-term weight loss, in addition to improving coexisting obesity-related conditions (e.g., obstructive sleep apnea, poorly-controlled type 2 diabetes, hypertriglyceridemia) (104). However, these diets require close metabolic monitoring (~at least every 2 weeks), and should only be prescribed and adjusted under the supervision of a physician specializing in obesity care. Medical contraindications include recent myocardial infarction, cardiac conduction disorders, history of cardiovascular disease, renal or hepatic disease, cancer, type 1 diabetes, and pregnancy. Behavioral contraindications to their use include bulimia nervosa, major depression, bipolar disorder, substance abuse, and acute psychiatric illness. The advantages of the VLCD for patients include a rapid improvement in blood pressure, blood glucose, serum lipids and often-psychological status. For those who require surgery, the rapid loss of weight may reduce some of the surgical risks associated with obesity.

8.3.2 Formulations Available

The hallmarks of the VLCD are the low calorie level and a relatively high percent of protein; which is at least 0.8 grams, but up to 1.5 grams per kilogram of ideal body weight (39;105). Protein needs are elevated on VLCD because in the hypocaloric state, the efficiency of protein utilization for maintaining the body’s lean cell mass is lessened since some of the amino acids are metabolized to produce glucose. Also, very heavy people who often are candidates for therapy have a larger lean body mass, and thus more lean tissue, as well as much more fat than their smaller peers. Even after adjustment for their greater fatness, total protein needs, which are most highly associated with the size of the lean body mass, are elevated. Therefore, higher protein levels may help to preserve protein nutritional status, although this remains to be demonstrated. VLCDs also have extremely low fat content and relatively low carbohydrate levels, making them ketogenic. Without special formulation or supplementation, the VLCD is deficient in several vitamins and minerals, specifically potassium, calcium, iron, zinc, vitamin C, vitamin B6, copper, and possibly other nutrients.

There are two major types of VLCDs currently in use; commercial and "home-made" preparations. The commercial preparations include powdered products that are rich in egg- or milk-based proteins, are mixed with water, and consumed four to five times per day. The commercial products must provide at least 70 grams of protein by law, and often contain much higher amounts of high quality protein (70 to 100 grams), 50 to 100 grams carbohydrate, and up to 15 grams fat per day, plus vitamins and minerals in amounts to meet the Recommended Dietary Allowances (RDA). These products are formulated under FDA regulatory specifications. They are convenient and have a predictable and adequate composition when used as directed. Their major disadvantage compared to home preparations is their higher cost. The formulas or prepackaged meals are relatively choice-free and help dieters avoid contact with conventional foods, which in some cases may facilitate dietary adherence and remove temptation.
Several commercial weight loss programs are available that provide an entire program of commercially prepared VLCDs plus the other essential aspects of a sound weight control program, including dietetic advice, exercise, behavioral modification, and supervision during the VLCD and post VLCD phases. The choices include the programs of HMR^TM (Health Management Resources), Optifast^TM (Novartis Nutrition), and Medifast^TM. These programs employ health professionals who are trained in weight management, and a structured program that encourages adherence. The major disadvantage is that they are expensive ($3,000 to $34,000 for 26 to 28 weeks), and costs may not be covered by health insurance (105). Also, there is the uncertainty that the weight that is lost will remain so over the long run. Therefore a serious psychological as well as economic investment of effort in long-term weight management is also mandatory. See Table 13 for available program details.

Table 13. Medically Supervised Meal Replacement Programs
Program/Product and Company	Description	Is product medically supervised?
HMR ^TM (Health Management Resources)	The HMR Decision-Free ^TM Clinic Weight-Loss Program: food provided includes shakes, puddings, soups, entrees, bars, and multigrain hot cereals.	Yes
Medifast ^TM	Provides special meal-plans for women, men, patients with diabetes, seniors, and teens. Six meals per day are prescribed, and foods offered include shakes, bars, soups, scrambled eggs, oatmeal, chili, puddings, and hot and cold drinks. All products are suitable for people with type 2 diabetes.	Yes
Optifast ^TM (Novartis Nutrition)	Comes as a powder to be mixed with water or as a liquid ready-to drink beverage. Patients are prescribed 5 packets of formula every 3-4 hours per day, in place of meals.	No

The "home made" VLCD regimens are sometimes referred to as "protein-sparing fasts", or "protein sparing modified fasts" (PSMF). This is a misnomer since they do not “spare” protein except in contrast to a total fast. They are usually based on lean meat, fish or poultry and a few other foods plus supplements of two to three grams of potassium chloride and a multivitamin/multimineral supplement in amounts approximating the Recommended Dietary Allowances (RDA). Without such supplementation, they may be nutritionally inadequate. When patients are provided with appropriate dietetic counseling and health supervision by a physician who is experienced in the use of VLCDs and other aspects of a complete weight reduction program, these formulations are also safe and generate rapid weight loss. The extremely hypocaloric versions of VLCDs (e.g., less than or equal to 800 calories per day), which are low in carbohydrate and sodium, promote a mild ketosis that gradually leads to diuresis and rapid weight loss in the first several days on the diet.

8.3.3 Use of Very-Low-Calorie Diets (VLCDs)

Evaluation of general health and cardiac status is important prior to the institution of a VLCD. Evaluation of medication dosages and physician monitoring during the regimen are also important, since with weight loss dosing may need to be adjusted. Many practitioners begin the regimen with a two to four week low calorie diet (LCD) phase to assess the ability to comply with a restrictive regimen, and to begin the weight loss process. This is followed by a 12 to 16 week VLCD phase; the regimen is limited to this amount of time to avoid excessive loss of lean tissue. The VLCD phase is then followed by a 12 to 14 week refeeding phase of transitioning back to usual foods and gradually increasing caloric levels. The goal is to increase calories from healthful foods up to 1,200 to 1,500 calories per day, increasing caloric intake by 100 to 150 calories per day (39). This helps to avoid rapid weight changes due to refeeding with restoration of glycogen stores and shifts in water balance. The refeeding phase also provides a time for assisting the dieter to plan a maintenance diet on conventional foods and to solidify a physical activity schedule. VLCD are most effective when administered as part of a more general weight control program that includes physical activity, nutrition education, behavioral modification and attention to decreasing other risk factors. If additional weight loss is needed, it is recommended that several months elapse before another VLCD phase is instituted (106). Although lean tissue is lost on most weight reduction diets, this is a particular risk on VLCD, since greater energy restrictions are associated with more lean tissue losses (73).

8.3.4 Safety of Very-Low-Calorie Diets (VLCDs)

The VLCD induces semi-starvation, which has both benefits and risks to the patient. Occasionally, with inadequate commercial products, such as one sold in the 1970’s that consisted of hydrolyzed collagen (an incomplete protein consisting solely of the amino acid glycine) with inadequate amounts of electrolytes, vitamins and minerals, deaths occurred (107). Today, commercial products are better regulated and are nutritionally complete by law; however, the potential for misuse still exists.
Some physiological effects are inevitable on VLCDs. On VLCDs mild ketosis occurs and increases risks of dehydration, although dehydration can be avoided by ample fluid intake. Patients on VLCDs should drink at least two liters of non-caloric liquids per day (preferably water) to make up for decreased food intake and to prevent dehydration. Avoidance of caffeinated beverages is sometimes recommended, as they can further the risk of dehydration, although moderate use is not prohibited (39). Electrolyte imbalances may occur, and so may nutrient deficiencies if measures are not taken to prevent them on "home-made" VLCD, by use of appropriate supplements. Minor side effects that occur, even with appropriate physician monitoring of cardiac and general health status, include fatigue, dizziness (due to orthostatic hypotension), muscle cramps, gastrointestinal distress (constipation and/or diarrhea), and cold intolerance. The risk of cholelithiasis (gallstones) is increased, and seems to be particularly high when weight loss is very rapid (e.g., >1.5 kilograms/week). The risk of cholelithiasis can be decreased by administering ursodeoxycholic acid, including a moderate amount of fat in the diet, and limiting the amount of weight loss to 1.5 kilograms per week (105).

8.3.5 Effectiveness of Very-Low-Calorie Diets (VLCDs)

Because these VLCDs are so low in energy, they usually produce a greater initial weight loss than LCDs. Patients who completed a comprehensive VLCD program including lifestyle modification lost an average of 15 to 25% of initial weight within three to four months (105). However, in comparisons of VLCDs with energy levels of approximately 800 calories versus diets at lower caloric levels of 400 to 500 calories, the lower VLCDs did not necessarily result in greater weight loss, perhaps because compensatory reductions in resting energy expenditure, discretionary physical activity, and the lack of adherence on the lower calorie regimes thwarted weight loss (108).

There seems to be little difference in outcomes between commercial and properly formulated homemade VLCDs. The NIH expert panel review of existing studies found that preservation of weight loss over the long-term (e.g., >1 year) was not different on VLCD from that of LCD since most patients gained back 30 to 50% of the lost weight. Studies of VLCDs vary in their long-term results, but weight regain is common (~3.1-3.7 kg during 21-38 weeks afterwards) (31). Combining a VLCD with behavior therapy, physical activity, and active physician follow-up may help to prevent this weight regain, and lend to greater weight loss (109). As such, the long-term advantages of VLCDs in weight control are unclear. Although weight gain is common after cessation of VLCDs, individual clinicians may decide that the expense and quick initial weight loss are worth it for the patient (39).

8.4 Fasting and Alternate Day Fasting

Total fasting is contraindicated for weight reduction because it causes excessive breakdown of lean tissue and ketosis. Also, the compensatory decreases in resting metabolism and physical activity on total fasts are profound and counterproductive, since they lower energy output (24;5).

Short-term modified alternate-day fasting (ADF) is a relatively new dietary strategy that has not yet received enough research attention to support the effectiveness of its use. On typical ADF diets, patients consume 25% of their energy needs on the fast day, and food intake ad libitum the next day. Many of the ADF studies do not include control groups that undertake other dietary approaches to weight loss, plus the sample sizes have been small (15-64 subjects) and the durations short (8-12 weeks) (110; 111; 112; 113; 114). However, results to date show similar weight loss and improvements in metabolic factors as compared to studies using other dietary approaches to weight loss, as well as good adherence. For example, Varady et al found that ADF was a viable diet option, helping obese patients not only to lose weight, but to also decrease their risk of coronary artery disease (CAD) (110). Emerging evidence suggests that the relative contribution of fat and carbohydrate in ADF diets does not have an impact on weight and blood lipid profiles (115). An alternative fasting regimen that has become popular among some dieters is two days of fast followed by five days of usual eat. Further research is needed with larger samples, dietary control groups, and longer durations before ADF’s widespread use for weight loss purposes, however.

An alternative intermittent fasting regimen that has become popular among some dieters is two non-consecutive days of fasting and five unrestrictive days of usual eating each week. This “5:2 Diet”, developed by Dr. Michael Mosley in the United Kingdom, has spread widely throughout Europe, and now the United States. For the two fast days, men are to eat no more than 600 kcals, and women 500 kcals. Usually this consists of a very light breakfast and dinner with little or no lunch. Anecdotally, weight loss has been similar to other dietary approaches to weight loss (~2 pounds/week) for short terms (~6 weeks). However, research is lacking on this specific type of diet, so its long-term safety and efficacy has not been tested, and its appropriateness in varying populations is currently unknown

9. Consider the Composition of the Reducing Diet

The composition of the reducing diet is important because it may influence both the composition of the weight that is lost and nutritional status. Several published overviews of some popular diets and the basic principles that must be considered in weight control can provide more information (72;91;102;116). Over the past several decades, the potential of varying dietary composition for purposes of weight loss has been studied extensively, yet not one universally optimal diet for all patients has emerged. “A variety of dietary approaches can produce weight loss in overweight and obese individuals” (31). The challenge for practitioners is to identify which diet would be most suited for each individual patient. This may be based on previous dieting experiences, personal food preferences, lifestyle, and other factors (57).

Dietary composition on reducing diets should be geared towards decreasing risks of nutrient inadequacy and diet-related chronic diseases. Accordingly, the diet should be adequate in all nutrients, to prevent deficiencies, while following dietary guidelines for health, performance (cognitive and physical), and well-being (24;117). Consumption of vegetables, legumes, fruits, whole grains, lean sources of protein, and water should be encouraged, with emphasis on balance and moderation (33). Diets that promote extreme restriction or unusually high intakes of any macronutrient or food should be limited to a short amount of time. Recommendations for healthful composition of weight reducing diets are outlined in the 2010 Dietary Guidelines for Americans (Table 1) and discussed further in this section.

Several studies have concluded that a reduced calorie diet results in clinically meaningful weight loss regardless of what macronutrients are emphasized (31, 118; 119). For example, in a study with 811 overweight adults placed on one of four diets, the best predictor or weight loss was dietary adherence. The targeted percentages of energy derived from fat, protein, and carbohydrates in the four diets were 20, 15, and 65%; 20, 25, and 55%; 40, 15, and 45%; and 40, 25, and 35%. After six months individuals lost an average of six kilograms, 7% of their initial body weight, and after 12 months they started to regain weight. At the end of the two-year study there were no differences in the amount of weight lost amongst participants. Changes from baseline differed among the different diet groups by less than 0.5 kilograms of body weight, and less than 0.5 centimeters at the waist. However, all of the diets reduced risk factors for cardiovascular disease and diabetes at both six months and two years (120). Similarly, a systematic review of long term randomized controlled trials comparing the Atkins, Weight Watchers, Zone, and South Beach diets showed similar modest weight loss with all four approaches [Atallah et al., 2014].

According to the most recent Guidelines for Managing Overweight and Obesity in Adults (31), which is supported by a systematic evidence review by several expert panels, clinically-meaningful weight loss can be achieved through various dietary strategies. The best predictor of weight loss is adherence to a diet that produces a negative caloric balance. Thus, practitioners and patients are challenged to work together to find the option that will best help the patient to adhere. The 15 dietary strategies identified by the expert panel are listed in the table below (31). They all produced caloric deficit, but either through prescribed calorie levels, through restriction or elimination of foods or food groups, or through targeting food groups or providing foods. The expert panel concluded that the strength of the evidence was high for these studies.

High protein (30%) Zone-type (40% carbohydrate) with 5 meals dailyEuropean Association for the Study of Diabetes GuidelinesLow carbohydrate (initially <20g/day)High protein (25%), moderated carbohydrates (45%)Low fat (10-25%) vegan-styleModerate protein (12%), higher carbohydrate (58%)Low fat (~20%)High or low glycemic load mealsLow glycemic load

Table 14: Fifteen dietary strategies identified by an expert panel as having sufficient empirical evidence to recommend for weight loss (31).
Prescribed Calorie Deficit^	Caloric Deficit through Restriction/Elimination	Caloric Deficit through Targeting Food Groups or Food Provision
Lacto-ovo-vegetarian	High protein (30%) Zone-type (40% carbohydrate) with 5 meals daily	European Association for the Study of Diabetes Guidelines
Low calorie	Low carbohydrate (initially <20g/day)	High protein (25%), moderated carbohydrates (45%)
Low glycemic load	Low fat (10-25%) vegan-style	Moderate protein (12%), higher carbohydrate (58%)
Low fat (<30%), high dairy & fiber	Low fat (~20%)	High or low glycemic load meals
Macronutrient targeted*	Low glycemic load
Mediterranean-style
AHA-style step 1
^Calorie prescription was either calculated or fixed, as described in section 8 *Macronutrients have ranged 15-25% protein, 20-40% fat, and 55-65% carbohydrate in these studies

9.1 Macronutrient Distribution

As described above, the macronutrient composition of the diet does not appear to play a major role in overall weight loss; reduced-calorie diets result in clinically meaningful weight loss regardless of which macronutrients they emphasize (121; 118). There is one exception: over the short term, low carbohydrate diets are ketogenic, and may cause a greater loss of body water than body fat (at least in the first few days of the diet). Water weight is regained when the diet ceases, or when carbohydrate intake increases. In this case, glycogen stores, which hold water, are regained. Generally, when any reducing diet is maintained over the long term, if it remains hypocaloric, it will result in a loss of body fat – regardless of the distribution of macronutrients.

Although weight loss is caused by many reduced-calorie diets, the nutritional adequacy of different calorie levels and macronutrient composition for weight loss diets varies (122). The lower the reducing diet is in calories, and the more its composition differs from usual levels, the greater the risk of nutrient inadequacy. For the most part, moderate fat, balanced macronutrient reduction diets are nutritionally adequate. Very low-fat diets tend to be deficient in vitamins E, B12, calcium, iron and zinc. High fat, low carbohydrate diets are nutritionally inadequate, and require supplementation to make them nutritionally adequate in many nutrients (96;123;124). They are often low in fiber, so constipation may occur. In this case, fiber supplements and ample water intake may be recommended. Dietary supplements used on weight reduction diets should be within RDA levels and below upper safe limits.

Metabolic parameters may improve on some various popular diets including decreased blood pressure, blood lipids, blood sugar, and serum insulin, related to energy restriction and weight loss, regardless of the macronutrient composition of the diet. However, there are some differences. Moderate fat, balanced nutrient reduction diets lower low-density lipoprotein (LDL) cholesterol, normalize plasma triglycerides, and normalize ratios of HDL/total cholesterol. High fat, very low carbohydrate diets result in ketosis. Low and very low-fat diets (e.g., 15-20% of calories) reduce low-density lipoprotein (LDL) cholesterol, and after a transient rise in triglycerides, may also decrease plasma triglyceride levels. Low carbohydrate diets (e.g., <100 grams of carbohydrate) that result in weight loss may also cause a decrease in blood lipids, blood glucose and insulin levels, and blood pressure. However, these diets are often high in saturated fat, total fat, and in dietary cholesterol, and low in plant-based nutrients such as fiber, so variability in metabolic responses may be seen, due in part to genetic predisposition. Moreover, these diets are ketogenic, often causing signs and symptoms such as diuresis, dizziness, halitosis, fatigue, weakness, hypotension, and malaise.

Hunger may vary on the different diets, and also from one individual to the next, but little objective evidence is available for comparing different reducing diets on their anti-hunger effects. Many factors affect hunger, appetite and subsequent food intake, including interactions between physiological and non-physiological factors. Schoeller and Buchholz speculate that a greater consumption of protein may increase satiety, which in turn results in better adherence to hypocaloric diets, however, substantial long-term evidence to support this supposition is lacking (124), and more research is needed in this area.

Long-term dietary adherence is likely to be a function primarily of psychological and lifestyle issues rather than macronutrient composition itself. At present little is known about the nutritional or other characteristics of diets that maximize adherence. It is likely that "one size does not fit all" in this respect, so the importance of individualization is underscored.

9.2 Protein

This section outlines dietary protein needs during weight reduction. The Recommended Dietary Allowance for protein is 0.8 grams per kilogram per day, but most Americans eat about 1.2 grams per kilogram per day, or approximately 15% of their total caloric intakes from protein. For people in energy balance and at a stable weight, the World Health Organization (WHO) recommends that dietary protein should account for approximately 10 to 15% of energy intake (125).

9.2.1 Protein Needs During Weight Reduction

Protein requirements tend to rise on hypocaloric diets, especially on VLCDs when protein is burned for energy. Thus, protein needs increase so that loss of lean body mass can be minimized. This is because when energy intakes are insufficient, glucogenic amino acids are used to maintain blood glucose levels and other ketogenic amino acids must be used for energy, so overall protein requirements increase. Fortunately, the hormonal milieu in hypocaloric states spares nitrogen to some extent and causes preferential use of fat for energy. However, fatty acids cannot be converted to blood glucose, so glucogenic amino acids are needed for this. Inevitably, as adipose tissue is mobilized some lean tissue is lost and consequently some nitrogen is also lost. Losses of water, calcium, phosphorus, potassium, and vitamins follow the loss of lean tissue. Excess losses of lean body mass can be hazardous, affecting cardiovascular function, exercise tolerance, and possibly immune responses, and thus should be avoided. As mentioned previously, excess loss of lean tissue can result from energy deficits that are too great (73).

As a rule of thumb, a minimum of 65 to 70 grams of protein is needed daily. On a VLCD, 1.5 grams of high quality protein per kilogram of ideal body weight per day is desirable, with intakes no less than, and preferably more than, 65 to 70 grams daily. Intakes may need to be even higher if the dieter suffers from certain diseases or is physically stressed, since nitrogen losses may be more extreme in these states. On diets providing 600 to 1,200 calories per day, daily protein intake should be at least one gram per kilogram ideal body weight per day. Reducing diets over 1,200 calories per day should supply at least 0.8 grams per kilogram ideal body weight, and more if the individual is physically active. Levels should remain this high after weight loss has stopped and maintenance has begun.

9.2.2 High Protein Weight Loss Diets

High protein reducing diets are those that provide more than 1.6 grams per kilogram of desirable weight per day. Self-prescribed high protein reducing diets vary in their composition from about 28 to 65% of energy, providing 71 to 163 grams of protein per day. They are currently popular as a new strategy for losing weight, and are usually quite low in their carbohydrate content. Some are clearly ketogenic, and severely limit carbohydrates to below 50 grams per day. Examples include the Doctor’s Quick Weight Loss Diet (126) The Dukan Diet (126), Dr. Atkins™’ Diet Revolution (127), The 17 Day Diet (231), and various iterations of the Paleo Diet, which is discussed in more detail below. Diets that are extremely high in protein should not be undertaken for long periods of time, since their long-term safety has not been sufficiently examined.

Other diets are extremely high in protein, very low in carbohydrate and ketogenic, but also very high in fat, such as Protein Power (128). Two other high protein diets with enough carbohydrate so that they are not likely to be ketogenic are The Zone (129) and Sugar Busters (130).

Many high protein diets include elaborate instructions that prescribe strict, structured eating schedules, and involve limited food variety and dietary flexibility. The high protein diets that are ketogenic also induce quick initial weight loss because of their low caloric level, and their diuretic effect owing to glycogen depletion, and sodium and water loss. They may also be associated with decreased appetite due to the high protein intake, since protein may show to be particularly satiating (131;132). Ketosis has long been said to reduce appetite, although little data supports this. Nonetheless, for some patients these constraints may help them to achieve and maintain low calorie intakes over the short run.

Popular high protein reducing regimens are not risk-free, however. Many of these diets advocate very high intakes of protein from meat and other foods that are also often high in saturated fat, cholesterol and sodium while they are low in dietary fiber, antioxidants, potassium, calcium, magnesium, and some vitamins. The purine content of meat, poultry, seafood, eggs, seeds, and nuts is high, and can increases uric acid levels and risk of gout in susceptible persons. The high protein load may also increase urinary calcium loss if it is not buffered (133). In patients with diabetic nephropathy, very high protein diets may speed progression, although the data are not definitive (134). Because many high protein diets are often by default low in carbohydrate, they also can cause an increase in ketosis. Finally, these diets do not necessarily promote greater long-term weight loss as compared to other options (135;136; 118).

9.3 Fat

This section outlines dietary fat needs during weight reduction.

9.3.1 Fat Needs During Weight Reduction

Even on reducing diets, the human body needs small amounts (e.g., three to six grams) of essential fatty acids (linoleic or arachidonic acid). Some fat is also necessary as a carrier for the fat-soluble vitamins A, D, E, and K. Therefore the diet should not be devoid of fat. However, because fat is calorically dense, it is often decreased on reducing diets to reduce energy intake while increasing bulk.

9.3.2 Moderate to Low-Fat Balanced Deficit Reducing Diets

In general, levels of dietary fat, saturated fat, trans fat, polyunsaturated fat, monounsaturated fat, and cholesterol should follow guidelines from the American Heart Association (AHA) on weight reduction diets. While lower levels may be appropriate in some cases, they amply meet requirements while supporting cardiovascular health (24).
Weight reduction diets that are moderate to low in fat (20 to 30% of calories) are called "balanced deficit" diets because they maintain a reasonable balance among macronutrients similar to that recommended in MyPlate, DASH, and the Dietary Guidelines for Americans (15; 117). They tend to achieve most of the caloric deficit by reducing fat from the typical level in North American Diets of about 34% or more of calories to 20 to 30% fat, 15% protein, and 55 to 65% of calories from carbohydrates. Some examples of balanced deficit diets are the Weight Watchers® Diet (25% fat, 20% protein, and 55% carbohydrate, with 26 grams of dietary fiber), Jenny Craig®, the National Cholesterol Education Program Step 1 diet (25% fat), diets based on the MyPlate, the DASH diet, the Shape up and Drop 10 diet of Shape Up! America (33), and the Nutrisystem® diet. Popular diet books using this approach include The Biggest Loser Diet (232), The Mayo Clinic Diet (233), and The Engine 2 Die (234). These dietary patterns have been extensively reviewed and appear to be effective for weight reduction on low calorie diets for most individuals.

9.3.3 Very Low-Fat Reducing Diets (<20% Fat Calories)

Very low-fat diets such as the Pritikin Diet (137), the Ornish Diet (138), and The Spark Solution Diet (235) have been advocated not only for weight reduction, but also for improving cardiovascular risk profiles. The Ornish Diet, which is very low in fat (13% of calories) and saturated fat, very high in carbohydrate (81% of calories) and very high in fiber (38 grams), is part of a program that includes nonsmoking, exercise and behavior modification. It was shown to reduce some cardiovascular risk factors in a limited long term study (138). For those who can adhere to the Ornish regime it may be helpful. However, it may not be appropriate for all populations, such as diabetics.

9.3.4 High Fat Diets for Weight Reduction (55 to 65% Fat)

High fat reducing diets are also usually low or very low in carbohydrate (<200 grams carbohydrate per day). Some current examples include Dr. Atkins™’ Diet Revolution (127), Protein Power (128), the Carbohydrate Addicts Diet (139), Dr. Bernstein’s Diabetes Solution (140), Life Without Bread (141), the Pennington Diet (141), and the Bulletproof Diet (236). There is some evidence that free-living, overweight people who self-select high fat, low carbohydrate diets consume fewer calories and lose weight (102). This is not because the laws of thermodynamics are violated, but because they have far fewer food options, if they adhere to such rigorous regimens. When high fat, low carbohydrate reducing diets are fed they also tend to cause ketosis and diuresis. They may also result in decreased blood lipids, glucose and insulin, along with and decreased blood pressure, but only if weight is lost. Over the short term (a few days or weeks) high fat, low carbohydrate, ketogenic diets cause a greater loss of body water than body fat, but water balance is quickly restored when carbohydrate intakes increase or when the diet ends. High fat, low carbohydrate diets are often nutritionally inadequate, so they often require some supplementation with micronutrients and fiber. If such high fat levels are continued on a chronic basis after weight is lost, they are may increase dietary risks for coronary artery disease. More research is needed in this area.

9.4 Carbohydrates and the Glycemic Index

The following section reviews carbohydrate needs during weight reduction, and the glycemic index.

9.4.1 Carbohydrate Needs in Weight Reduction

Carbohydrate needs for most individuals are at least 50 grams per day, to fuel the central nervous system, red blood cells, and other glucose-dependent tissues. If carbohydrate intake fall below this, than gluconeogenesis is likely to ensue. The carbon source for gluconeogenesis cannot be fatty acids, so in these cases, amino acids are used to maintain blood glucose and fuel glucose-dependent tissues. At least 100 grams carbohydrate, and preferably carbohydrate within the Acceptable Macronutrient Distribution Ranges (AMDR) of 45 to 65% of total energy intake, should be provided for diets that are over 800 calories per day. Under experimental conditions, both hypocaloric diets very high in sugars (mono-and di-saccharides) and diets very high in starches (digestible polysaccharides) that are equi-caloric have similar weight loss effects (143;144). However, from the practical standpoint, since many products that are high in sugar are calorically dense and often are also high in calories, added fat, and low in fiber, vitamins and minerals; sugars are usually limited on reducing diets. Additionally, high sugar diets may increase some cardiovascular risk factors (145). From the physiological standpoint, “added” sugar, and sugar inherently in the foods, are similar in their caloric contributions.
.
Individuals assigned to a low-carbohydrate, high-protein diets do lose more weight at six months than those on low-fat, reduced-energy diet. However, this difference is no longer significant at 12 months (70;116;120). An evidence review from the Academy of Nutrition and Dietetics concluded that, “An individualized, reduced calorie diet is the basis of the dietary component of a comprehensive weight management program. Reducing dietary fat and/or carbohydrates is a practical way to create a caloric deficit of 500 to 1,000 kcal below estimated energy needs and should result in a weight loss of one to two pounds per week” (70). Whether an individual dieter reduces fat or carbohydrate does not matter. If calories are similar, in the long run, weight loss amounts to reducing caloric intake. Concerns regarding an increase in cardiovascular risks with low-carbohydrate diets now do not appear to be as problematic as first thought (70). If an obese person loses weight, cardiovascular risk factors usually improve (31).

9.4.2 Low Carbohydrate Diets (<100 grams Carbohydrate)

Diets providing less than 100 grams of carbohydrate per day, and especially those with less than 50 grams carbohydrates per day, are ketogenic. Ketosis can be a problem on some popular diets that are very low in carbohydrates, such as Dr. Atkins™’ Diet Revolution (127), Protein Power (128), The Dukan Diet (126), Dr. Bernstein’s Diabetes Solution (140), The Bulletproof Diet (236),, and the Pennington Diet (142). Also, VLCDs containing fewer than 100 grams of carbohydrate per day are ketogenic and may lead to excessive protein breakdown to maintain blood glucose levels unless protein intakes are increased. When the body must rely on catabolism of protein to preserve blood glucose levels via gluconeogenesis, the catabolism of the protein is accompanied by loss of water. For every gram of protein (or glycogen) that is broken down, three grams of water are released, causing rapid weight loss but also a state of relative dehydration (95). Relative dehydration caused by ketosis and failure to drink adequate amounts of fluids is not only undesirable for health reasons, it reduces exercise tolerance (67). It also does not address the primary purpose of the weight-reducing strategy, which is to decrease excess adipose tissue and not water weight. One main concern with studies to date on low carbohydrate diets is that most of them to not include exercise. Since exercise tolerance declines with low carbohydrate intakes and reduced glycogen stores, more research is needed in this area before such diets can be recommended.

9.4.3 Low Glycemic Index Diets

The Glycemic Index (GI) was originally developed for the therapy of diabetes, but it has gained popularity in weight management. The GI describes the blood glucose response resulting from consumption of a defined amount of carbohydrate (usually 50 grams) from a given food, relative to the response of the same amount of carbohydrate from a control food (usually white bread)(146). In brief, the GI is an alternative system for classifying carbohydrate-containing foods according to their postprandial blood glucose responses to portions containing standardized amounts of carbohydrates (30). Since the GI is based on standardized portions, glycemic load (GL), the product of GI and carbohydrate amount, is used to evaluate the effect of meals/snacks—differing in both quality and quantity of carbohydrates—on postprandial glycemia (30).

The basic premise is that more moderate blood glucose and metabolic responses from low-GI foods and a low GL will sustain satiety and energy balance to a greater extent than would high-GI foods and a high GL load. The GI may be important in regulating hunger, voluntary energy intake, and satiety. A high-GI meal or snack may compromise glucose uptake following a subsequent meal—a phenomenon known as the “second-meal effect.” The underlying mechanism likely involves decreased insulin sensitivity with increased concentrations of circulating free fatty acids during the late postprandial phase. With regard to a high-GI meal or snack, it is thought that, “the drop in blood glucose during the middle postprandial phase may increase the preference for high-GI foods, leading to repeated cycles of excess hunger followed by hyperphagia that may last for several hours following restoration of euglycemia. These vicious cycles, exacerbated by the second-meal effect may contribute to disappointing long-term weight control with conventional low-fat diet prescriptions that emphasize the importance of consuming starchy foods” (30). Hence, low-GI foods are thought to help minimize blood glucose fluctuations, hunger hormones, and increase satiety.

Some, but not all, studies have conducted a follow-up period at 12 months showing that overweight or obese individuals on low-GI diets lose slightly more weight (1-3 kg) than those on high-GI diets or conventional energy restricted weight loss diets (147). Beyond short term weight loss, low-GI diets have also shown to decrease fasting glucose and insulin levels, reduce circulating triglycerides, and improve blood pressure (30;148;149). Thus, low-GI and low-GL diet plans may help some individuals lose weight, typically one to two BMI units, and help improve metabolic parameters and risk of cardiovascular disease. (148). The effects of low GI carbohydrates may also help to prevent excess weight gain, although more research must be done on their longer-term efficacy (150-152).

Consumption of whole grains, legumes, fruits, vegetables, and whole foods that are low in GI, is helpful in meeting fiber goals and may be helpful in weight management. A well balanced, hypocaloric low glycemic index diet may prove to be effective in properly educated, adherent patients who are willing to take the time to learn about high- and low-glycemic foods, and who do not completely exclude healthful high glycemic foods. For example, sausages, ice cream, and chocolate cake with frosting are all low GI foods, while parsnips, carrots, bananas, dates and potatoes tend to be high GI foods. This underscores the point that more than just GI must be considered in food choices and patients need to be educated accordingly. Since GI is not listed on food labels in most countries, and since many factors influence it, such as cooking, ripeness, and the other foods consumed at the same meal, this dietary approach may pose a challenge for some patients. Any reducing diet must be viewed as a whole. In the USA nutrient fact labels are available on most processed foods, and provide information on carbohydrate content. Therefore the GI does not offer much additional information. However labeling of carbohydrate subtypes is not done in some countries, and use of the GI is popular. As seen in the table at the beginning of this section, diets with altered GI have been applied in cases where calories were prescribed, where food was provided, and where intake was limited, and all produced similar results to other dietary approaches. However, data are limited in US populations. In summary, diets based on the GI may offer some patients benefits in terms of short and long-term weight loss, but the perpetuity of the regimen remains in question.

9.4.4 Paleo Diet

The Paleo Diet has gained considerable popularity among consumers in recent years, although no large-scale scientific study has thoroughly investigated it yet, especially for the purposes of weight loss. This dietary approach suggests that individuals consume only foods and beverages that presumably made up the diets of Paleolithic humans (153). Thus, all processed foods and beverages are eliminated in addition to grains, dairy, and legumes. Allowed foods include meats, fish, poultry, vegetables, fruits, and nuts (not peanuts). Deficits in caloric intake are achieved through elimination of large amounts of usually-consumed food types. Advantages are that this diet tends to be high in nutrients that come from vegetables and fruits (e.g. vitamin C), and it is low in sodium and glycemic index / load (154). However, it is low in calcium, and high in cholesterol (154). While it provides satiety, many subjects find adherence difficult, due to the restriction of so many foods and beverages. To date, most studies have only tested this diet short term (10 days to 3 months), with low subject numbers (9-20), sometimes lacking control groups, and without a concurrent exercise prescription (155; 154; 156; 157). Weight loss has been comparable to other similar dietary approaches (156), and improvements have been noted for several risk factors of cardiovascular disease and type 2 diabetes (154; 157), even without weight loss (155). Patients attempting this diet while on warfarin treatment should consult their physician or dietitian due to the high vitamin K coming from the abundance of vegetables (153). More work is needed to determine long-term outcomes and adherence for this diet. Particular attention should be given to bone and gastrointestinal health, since dairy, cereal fibers, and legumes are missing from this diet [158; 159; 160).

9.4.5 High-Fructose Corn Syrup and Weight

High-fructose corn syrup (HFCS) does not contribute to overweight or obesity any differently than do other energy sources (161). HFCS has been blamed for the obesity epidemic mainly due to the association between American’s increases in weight along with the increase in HFCS in our food supply since the 1970s, but similar associations are also present with bottled water sales. However, association is not causation. In 2004, Bray et al (162) hypothesized that HFCS was a direct causative factor for obesity. However, to date, there is no scientific evidence supporting this theory. As stated in White’s article from The American Journal of Clinical Nutrition, “The HFCS-obesity hypothesis of Bray et al relies heavily on the positive association between increasing HFCS use and obesity rates in the United States. However, Bray et al treated this association in isolation, offering no perspective on trends in total caloric intake or added sweeteners use in comparison with use of other dietary macronutrients.”

HFCS was introduced to the food industry in the late 1960s and was well received because it is stable in acidic foods and beverages, is easily transportable and is sweet. HFCS can be pumped from delivery vehicles to storage and mixing tanks, requiring only simple dilution before use. Furthermore, it has remained relatively inexpensive. Its sweetness mirrored that of sugar. Contrary to popular belief, HFCS is not sweeter than sucrose. The forms of HFCS in the food supply are HFCS-55 and HFCS-42 with 55% fructose and 42% glucose, and 42% fructose and 53% glucose, respectively. The remaining carbohydrates are free glucose, maltose, and maltotriose. A similar ratio of fructose to glucose as in HFCS is also in honey, invert sugar, fruit, and fruit juices (163). Table sugar or sucrose is composed of 50% fructose and 50% glucose. Hence, the ratio of glucose to fructose in both HFCS and sucrose is essentially 1 to 1. Furthermore, HFCS and sucrose both contain four calories per gram. Existing theoretical and empirical evidence suggests that fructose-induced problems are not more related to HFCS than sucrose intake (164). Total caloric intake is positively associated with BMI, independent of sugar intake (165). However, the World Health Organization and U.S. Dietary Guidelines recommend avoidance of excess added sugars due to their ‘empty calories’, which may dilute dietary quality, and also may elevate some cardiometabolic risk factors (117; 145),
HFCS is not a direct cause of the obesity epidemic in the United States. To date, there is no evidence linking these two factors (161;166;167). As Forshee et al concluded, “Evidence from ecological studies linking HFCS consumption with rising BMI rates is unreliable. Evidence from epidemiologic studies and randomized controlled trials is inconclusive. Based on the currently available evidence, an expert panel concluded that HFCS does not appear to contribute to overweight and obesity any differently than do other energy sources” (161).

9.5 Water

This section discusses water and electrolyte needs during weight reduction.

9.5.1 Water Needs on Reducing Diets Vary

Ample fluid intake is important on weight reduction diets to prevent dehydration, especially if diets are ketogenic, very low in calories, or being undertaken in hot climates or with physical exertion. As mentioned earlier, losses of body glycogen and protein are accompanied by losses of body water. Intake of low-calorie or calorie-free fluids such as water should be emphasized (168). Water needs go up with increases in physical activity, not only due to sweat losses, but also due to increased water losses due to respiration (67). The fatigue that some dieters associate with hypocaloric diets is often due in part to dehydration, especially if they have also increased their physical activity and exercise regimes dramatically. Body water losses of as little as 2% have been associated with decreased physical and mental performance, and impaired thermoregulation (168). Some dieters may be in a state of mild dehydration much of the time, and this is unnecessary and may detract from quality of life. General water recommendations average approximately 2.7 liters (91 ounces) per day for women and 3.7 liters (125 ounces) per day for men. This includes total water intake from all beverages and water in foods. It has been estimated that 20% of total water consumption comes from solid foods (169). A fluid intake plan should be incorporated in every weight loss regimen. Non-caloric sources of fluid should be emphasized; pure water and seltzers tend to be great choices.

9.6 Electrolytes

Under normal circumstances on a well-balanced diet that is not overly restrictive with energy, electrolyte balance is maintained. If an individual may be losing excess electrolytes due to high sweat or urine losses, electrolytes can usually be replaced with normal foods (67). The American diet is overly abundant in sodium. Potassium is not so abundant but can be obtained in fruits and vegetables. Examples of foods that are high in both sodium and potassium include tomato sauces and vegetable soups.
Electrolyte levels are of particular concern on VLCD, since occasionally cardiac arrhythmias have resulted from hypokalemia on such regimens (105). Since hypokalemia can be fatal, electrolyte levels must always be monitored on VLCD.

9.7 Vitamins and Minerals

The next section outlines vitamin and mineral needs during weight reduction.

9.7.1 Vitamin and Mineral Needs During Weight Reduction

Vitamin and mineral nutrition is critical during weight reduction and maintenance. The amounts of nutrients specified in Recommended Dietary Allowance (RDA) for an individual’s age and sex must continue to be met, even on reducing diets for all other nutrients (See Table 15 and Table 16). The lower the diet is in calories, the more likely it is that essential vitamins, minerals and electrolytes such as potassium, copper, magnesium, Vitamin E, Vitamin B6, folic acid, iron, and calcium are likely to be low. As a rule of thumb, diets below 1,200 calories per day are likely to require vitamin and mineral supplements in amounts approximating the Recommended Dietary Allowances (15). Above 1,200 calories per day, women in reproductive age groups may still need iron, calcium, and folic acid supplements, since their needs for these nutrients are high, but most other nutrient needs can be met by a well-balanced diet that follows the Dietary Guidelines for Americans (15). For this reason, foods with high micronutrient density, but low energy density are especially important to include on a reducing diet. They include fruits, vegetables, legumes, and lightly processed whole grains. Table 15, Table 16, Table 17, and Table 18 present the current DRIs for vitamins, minerals and tolerable upper levels (UL) for these same nutrients.

Table 15.
Dietary Reference Intakes (DRIs): Recommended Intakes for Individuals, Vitamins (292)
Food and Nutrition Board, Institute of Medicine, The National Academies

Life Stage
Group

Vitamin A

Vitamin C

Vitamin D ^±

Vitamin E

Vitamin K

Thiamin

Riboflavin

Niacin

VitaminB6

Folate

Vitamin B12

Pantothenic Acid

Biotin

Choline

(µg/d) ^a

(mg/d)

(µg/d) ^b,c

(mg/d) ^d

(µg/d)

(mg/d)

(mg/d) ^e

(mg/d)

(µg/d) ^f

(µg/d)

Acid (mg/d)

(µg/d)

(mg/d) ^g

Infants

0–6 mo

400*

40*

2.0*

0.2*

0.3*

0.1*

65*

0.4*

1.7*

125*

7–12mo

500*

50*

2.5*

0.3*

0.4*

0.3*

80*

0.5*

1.8*

150*

Children

1–3 y

300

30*

0.5

150

0.9

200*

4–8 y

400

55*

0.6

200

1.2

12*

250*

Males

9–13 y

600

60*

0.9

1.0

300

1.8

20*

375*

14–18 y

900

75*

1.2

1.3

400

2.4

25*

550*

19–30 y

900

120*

1.2

1.3

400

2.4

30*

550*

31–50 y

900

120*

1.2

1.3

400

2.4

30*

550*

51–70 y

900

10*

120*

1.2

1.3

1.7

400

2.4 ^h

30*

550*

> 70 y

900

15*

120*

1.2

1.3

1.7

400

2.4 ^h

30*

550*

Females

9–13 y

600

60*

0.9

1.0

300

1.8

20*

375*

14–18 y

700

75*

1.0

1.2

400 ⁱ

2.4

25*

400*

19–30 y

700

90*

1.1

1.3

400 ⁱ

2.4

30*

425*

31–50 y

700

90*

1.1

1.3

400 ⁱ

2.4

30*

425*

51–70 y

700

10*

90*

1.1

1.5

400

2.4 ^h

30*

425*

> 70 y

700

15*

90*

1.1

1.5

400

2.4 ^h

30*

425*

Pregnancy

≤ 18 y

750

75*

1.4

1.9

600 ^j

2.6

30*

450*

19–30 y

770

90*

1.4

1.9

600 ^j

2.6

30*

450*

31–50 y

770

90*

1.4

1.9

600 ^j

2.6

30*

450*

Lactation

≤ 18 y

1,200

115

75*

1.4

1.6

2.0

500

2.8

35*

550*

19–30 y

1,300

120

90*

1.4

1.6

2.0

500

2.8

35*

550*

31–50 y

1,300

120

90*

1.4

1.6

2.0

500

2.8

35*

550*

NOTE: This table (taken from the DRI reports, see www.nap.edu) presents Recommended Dietary Allowances (RDAs) in bold type and Adequate Intakes (AIs) in ordinary type followed by an asterisk (*). RDAs and AIs may both be used as goals for individual intake. RDAs are set to meet the needs of almost all (97 to 98 percent) individuals in a group. For healthy breastfed infants, the AI is the mean intake. The AI for other life stage and gender groups is believed to cover needs of all individuals in the group, but lack of data or uncertainty in the data prevent being able to specify with confidence the percentage of individuals covered by this intake.^a As retinol activity equivalents (RAEs). 1 RAE = 1 µg retinol, 12 µg ß-carotene, 24 µg ∂-carotene, or 24 µg ß-cryptoxanthin. To calculate RAEs from REs of provitamin A carotenoids in foods, divide the REs by 2. For preformed vitamin A in foods or supplements and for provitamin A carotenoids in supplements, 1 RE = 1 RAE.^b calciferol. 1 µg calciferol = 40 IU vitamin D.^c In the absence of adequate exposure to sunlight.^d As ∂-tocopherol. ∂-Tocopherol includes RRR- ∂ - tocopherol, the only form of ∂-tocopherol that occurs naturally in foods, and the 2R -stereoisomeric forms of ∂-tocopherol ( RRR -, RSR -, RRS -, and RSS -∂-tocopherol) that occur in fortified foods and supplements. It does not include the 2S -stereoisomeric forms of ∂-tocopherol ( SRR -, SSR -, SRS -, and SSS -∂-tocopherol), also found in fortified foods and supplements.^e As niacin equivalents (NE). 1 mg of niacin = 60 mg of tryptophan; 0–6 months = preformed niacin (not NE).^f As dietary folate equivalents (DFE). 1 DFE = 1 µg food folate = 0.6 µg of folic acid from fortified food or as a supplement consumed with food = 0.5 µg of a supplement taken on an empty stomach.^g Although AIs have been set for choline, there are few data to assess whether a dietary supply of choline is needed at all stages of the life cycle, and it may be that the choline requirement can be met by endogenous synthesis at some of these stages.^h Because 10 to 30 percent of older people may malabsorb food-bound B ₁₂ , it is advisable for those older than 50 years to meet their RDA mainly by consuming foods fortified with B ₁₂ or a supplement containing B ₁₂ .^± In 2008, the American Academy of Pediatrics adjusted their 2003 recommendations for vitamin D in children from 5 µg per day (200 IU), beginning in the first two months of life, to 10 µg per day (400 IU) within the first few days of life. This increased recommendation is based on the amount of vitamin D that can be given safely per day to prevent or treat rickets and possibly provide additional health benefits. The 2004 DRIs have not yet been updated to reflect this.ⁱ In view of evidence linking inadequate folate intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 µg from supplements or fortified foods in addition to intake of food folate from a varied diet.^j It is assumed that women will continue consuming 400 µg from supplements or fortified food until their pregnancy is confirmed and they enter prenatal care, which ordinarily occurs after the end of the periconceptional period—the critical time for formation of the neural tube.Copyright 2004 by the National Academy of Sciences. All rights reserved. 2/15/01

Table 16. Dietary Reference Intakes (DRIs): Recommended Intakes for Individuals, Elements (130)Food and Nutrition Board, Institute of Medicine, National Academies

Life StageGroup

Calcium

Chromium

Copper

Fluoride

Iodine

Iron

Magnesium

Manganese

Molybdenum

Phosphorus

Selenium

Zinc

(mg/d)

(µg/d)

(mg/d)

(µg/d)

(mg/d)

(µg/d)

(mg/d)

(µg/d)

(mg/d)

Infants

0–6 mo

210*

0.2*

200*

0.01*

110*

0.27*

30*

0.003*

100*

15*

7–12 mo

270*

5.5*

220*

0.5*

130*

75*

0.6*

275*

20*

Children

1–3 y

500*

11*

340

0.7*

1.2*

460

4–8 y

800*

15*

440

130

1.5*

500

Males

9–13 y

1,300*

25*

700

120

240

1.9*

1,250

14–18 y

1,300*

35*

890

3 *

150

410

2.2*

1,250

19–30 y

1,000*

35*

900

150

400

2.3*

700

31–50 y

1,000*

35*

900

150

420

2.3*

700

51–70 y

1,200*

30*

900

150

420

2.3*

700

> 70 y

1,200*

30*

900

150

420

2.3*

700

Females

9–13 y

1,300*

21*

700

120

240

1.6*

1,250

14–18 y

1,300*

24*

890

150

360

1.6*

1,250

19–30 y

1,000*

25*

900

150

310

1.8*

700

31–50 y

1,000*

25*

900

150

320

1.8*

700

51–70 y

1,200*

20*

900

150

320

1.8*

700

> 70 y

1,200*

20*

900

150

320

1.8*

700

Pregnancy

≤ 18 y

1,300*

29*

1,000

220

400

2.0*

1,250

19–30 y

1,000*

30*

1,000

220

350

2.0*

700

31–50 y

1,000*

30*

1,000

220

360

2.0*

700

Lactation

≤ 18 y

1,300*

44*

1,300

290

360

2.6*

1,250

19–30 y

1,000*

45*

1,300

290

310

2.6*

700

31–50 y

1,000*

45*

1,300

290

320

2.6*

700

NOTE: This table presents Recommended Dietary Allowances (RDAs) in bold type and Adequate Intakes (AIs) in ordinary type followed by an asterisk (*). RDAs and AIs may both be used as goals for individual intake. RDAs are set to meet the needs of almost all (97 to 98 percent) individuals in a group. For healthy breastfed infants, the AI is the mean intake. The AI for other life stage and gender groups is believed to cover needs of all individuals in the group, but lack of data or uncertainty in the data prevent being able to specify with confidence the percentage of individuals covered by this intake.SOURCES : Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B ₆ , Folate, Vitamin B ₁₂ , Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001). These reports may be accessed via www.nap.edu.Copyright 2001 by the National Academy of Sciences. All rights reserved. 2/15/01

Table 17. Dietary Reference Intakes (DRIs): Tolerable Upper Intake Levels (UL ^a ) for Vitamins (130)Food and Nutrition Board, Institute of Medicine, National Academies

Life Stage Group

Vitamin A

Vitamin C

Vitamin D

Vitamin E

Vitamin K

Thiamin

Riboflavin

Niacin

Vitamin B ₆

Folate

Vitamin B ₁₂

Pantothenic Acid

Biotin

Choline

Carotenoids ^e

(µg/d) ^b

(mg/d)

(µg/d)

(mg/d) ^c,d

---

(mg/d) ^d

(mg/d)

(µg/d) ^d

---

(g/d)

---

Infants

0-6 mo

600

ND ^f

7-12 mo

600

Children

1-3 y

600

400

200

300

1.0

4-8 y

900

650

300

400

1.0

Males, Females

9-13 y

1,700

1,200

600

2.0

14-18 y

2,800

1,800

800

3.0

19-70 y

3,000

2,000

1,000

100

1,000

3.5

> 70 y

3,000

2,000

1,000

100

1,000

3.5

Pregnancy

≤ 18 y

2,800

1,800

800

3.0

19-50 y

3,000

2,000

1,000

100

1,000

3.5

Lactation

≤ 18 y

2,800

1,800

800

3.0

19-50 y

3,000

2,000

1,000

100

1,000

3.5

^a UL = The maximum level of daily nutrient intake that is likely to pose no risk of adverse effects. Unless otherwise specified, the UL represents total intake from food, water, and supplements. Due to lack of suitable data, ULs could not be established for vitamin K, thiamin, riboflavin, vitamin B ₁₂ , pantothenic acid, biotin, or carotenoids. In the absence of ULs, extra caution may be warranted in consuming levels above recommended intakes.^b As preformed vitamin A only.^c As ∂-tocopherol; applies to any form of supplemental ∂-tocopherol.^d The ULs for vitamin E, niacin, and folate apply to synthetic forms obtained from supplements, fortified foods, or a combination of the two.^e ß-Carotene supplements are advised only to serve as a provitamin A source for individuals at risk of vitamin A deficiency.^f ND = Not determinable due to lack of data of adverse effects in this age group and concern with regard to lack of ability to handle excess amounts. Source of intake should be from food only to prevent high levels of intake.SOURCES : Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B ₆ , Folate, Vitamin B ₁₂ , Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001). These reports may be accessed via www.nap.edu.Copyright 2001 by the National Academy of Sciences. All rights reserved. 2/15/01

Table 18. Dietary Reference Intakes (DRIs): Tolerable Upper Intake Levels (UL ^a ), Elements (130)Food and Nutrition Board, Institute of Medicine, National Academies
Life Stage Group	Arsenic ^b	Boron	Calcium	Chrom-ium	Copper	Fluoride	Iodine	Iron	Magn-esium	Manga-nese	Molyb-denum	Nickel	Phos-phorus	Selenium	Silicon ^d	Van-adium	Zinc
Life Stage Group	---	(mg/d)	(g/d)	---	(µg/d)	(mg/d)	(µg/d)	(mg/d)	(mg/d) ^c	(mg/d)	(µg/d)	(mg/d)	(g/d)	(µg/d)	---	(mg/d) ^e	(mg/d)
Infants
0-6 mo	ND ^f	ND	ND	ND	ND	0.7	ND	40	ND	ND	ND	ND	ND	45	ND	ND	4
7-12 mo	ND	ND	ND	ND	ND	0.9	ND	40	ND	ND	ND	ND	ND	60	ND	ND	5
Children
1-3 y	ND	3	2.5	ND	1,000	1.3	200	40	65	2	300	0.2	3	90	ND	ND	7
4-8 y	ND	6	2.5	ND	3,000	2.2	300	40	110	3	600	0.3	3	150	ND	ND	12
Males, Females
9-13 y	ND	11	2.5	ND	5,000	10	600	40	350	6	1,100	0.6	4	280	ND	ND	23
14-18 y	ND	17	2.5	ND	8,000	10	900	45	350	9	1,700	1.0	4	400	ND	ND	34
19-70 y	ND	20	2.5	ND	10,000	10	1,100	45	350	11	2,000	1.0	4	400	ND	1.8	40
> 70 y	ND	20	2.5	ND	10,000	10	1,100	45	350	11	2,000	1.0	3	400	ND	1.8	40
Pregnancy
≤ 18 y	ND	17	2.5	ND	8,000	10	900	45	350	9	1,700	1.0	3.5	400	ND	ND	34
19-50 y	ND	20	2.5	ND	10,000	10	1,100	45	350	11	2,000	1.0	3.5	400	ND	ND	40
Lactation
≤ 18 y	ND	17	2.5	ND	8,000	10	900	45	350	9	1,700	1.0	4	400	ND	ND	34
19-50 y	ND	20	2.5	ND	10,000	10	1,100	45	350	11	2,000	1.0	4	400	ND	ND	40
^a UL = The maximum level of daily nutrient intake that is likely to pose no risk of adverse effects. Unless otherwise specified, the UL represents total intake from food, water, and supplements. Due to lack of suitable data, ULs could not be established for arsenic, chromium, and silicon. In the absence of ULs, extra caution may be warranted in consuming levels above recommended intakes.^b Although the UL was not determined for arsenic, there is no justification for adding arsenic to food or supplements.^c The ULs for magnesium represent intake from a pharmacological agent only and do not include intake from food and water.^d Although silicon has not been shown to cause adverse effects in humans, there is no justification for adding silicon to supplements.^e Although vanadium in food has not been shown to cause adverse effects in humans, there is no justification for adding vanadium to food and vanadium supplements should be used with caution. The UL is based on adverse effects in laboratory animals and this data could be used to set a UL for adults but not children and adolescents.^f ND = Not determinable due to lack of data of adverse effects in this age group and concern with regard to lack of ability to handle excess amounts. Source of intake should be from food only to prevent high levels of intake.SOURCES : Dietary Reference Intakes for Calcium, Phosphorous, Magnesium, Vitamin D, and Fluoride (1997); Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B ₆ , Folate, Vitamin B ₁₂ , Pantothenic Acid, Biotin, and Choline (1998); Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000); and Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc (2001). These reports may be accessed via www.nap.edu.Copyright 2001 by the National Academy of Sciences. All rights reserved. 2/15/01

9.7.2 Calcium Supplementation, Dietary Dairy Intake, and Weight Loss

Some studies in the past have suggested that calcium supplementation and/or supplementation of dairy products in the diet play a direct role in the prevention and treatment of obesity. However, not all data support this hypothesis, and several studies found that calcium or dairy consumption do not aid in weight loss (170), nor does calcium supplementation have an effect in preventing weight gain (171-173).

In a review evaluating 49 randomized clinical trials assessing the effect of dairy product or calcium supplement consumption, 41 studies showed no effect, two reported weight gain, one showed a lower rate of gain, and five showed it was effective as an aide in weight loss (170).

Major et al found that calcium plus vitamin D supplementation enhanced the beneficial effect of weight loss on the lipid profile; however, it had no effect on weight itself (174). There is also some evidence that high calcium or high dairy intakes during weight loss spare lean tissue loss to a greater extent than lower levels, although the evidence is not conclusive at this time.

9.7.3 Dietary Supplements and Weight Loss

Currently over half of the adult population uses dietary supplements (175). Most reported the motivation for using them was to increase over health even though less than a quarter of those supplements taken were recommended by a physician or healthcare provider (175). Retail sales of weight-loss supplements were estimated to be over $25.177 billion in 2008, including meal replacements (176).

Ingredient	Purported Mechanism	Research Findings	Safety+
Table 19.Common Ingredients in Weight Loss Dietary Supplements* (294)
Bitter orange (synephrine)	Increased energy expenditure and lipolysis; mild appetite suppressant	Small clinical trials of poor methodological quality demonstrating possible effect on resting metabolic rate and energy expenditure, with inconclusive effects on weight loss	Reported adverse effects include chest pain, anxiety, and increased blood pressure and heart rate
Caffeine (as added caffeine or from guarana, kola nut, yerba mate, or other herbs)	Stimulation of central nervous system; increased thermogenesis and fat oxidation	Short-term clinical trials of combination products showing possible modest effect on body weight or decreased weight gain over time	Safety concerns not usually reported at doses less than 400 mg/day for adults, but there are significant safety concerns at higher doses. Reported adverse effects include nervousness, jitteriness, vomiting, and tachycardia
Calcium	Increased lipolysis and fat accumulation; decreased fat absorption	Several large clinical trials have shown no effect on body weight, weight loss, or prevention of weight gain	No safety concerns reported at recommended intakes, but constipation, kidney stones, and interference with zinc and iron absorption can occur at intakes above 2,000–2,500 mg for adults
Chitosan	Binding of dietary fat in the digestive tract	Small clinical trials, mostly of poor methodological quality, have shown minimal effect on body weight	Reported adverse effects include flatulence, bloating, constipation, indigestion, nausea, and heartburn
Chromium	Increased lean muscle mass; promotion of fat loss; reduced food intake, hunger levels, and fat cravings	Several clinical trials of varying methodological quality have found minimal effect on body weight and body fat	Reported adverse effects include headache, watery stools, constipation, weakness, vertigo, nausea, vomiting, and urticaria (hives) when taken above recommended intakes (25–45 mcg/day for adults)
Coleus forskohlii (forskolin)	Enhanced lipolysis; reduced appetite	A small number of clinical trials show no effect on body weight	Unknown
Conjugated linoleic acid	Promotion of apoptosis in adipose tissue	Several clinical trials have shown minimal effect on body weight and body fat	Reported adverse effects include abdominal discomfort and pain, constipation, diarrhea, loose stools, dyspepsia, and possible adverse effects on blood lipid profiles
Ephedra (ma huang, ephedrine)	Stimulation central nervous system; increased thermogenesis; reduced appetite	Several short-term clinical trials of good methodological quality, many of ephedra combined with caffeine, have found modest effect on short-term weight loss	Banned as a dietary supplement ingredientReported adverse effects include anxiety, mood changes, nausea, vomiting, hypertension, palpitation, stroke, seizures, heart attack, and death
Garcinia cambogia (hydroxycitric acid)	Inhibited lipogenesis; suppressed food intake	Several short-term clinical trials of varying methodological quality have found little to no effect on body weight	Reported adverse effects include headache, nausea, upper respiratory tract symptoms, and gastrointestinal symptoms
Glucomannan	Increased feelings of satiety and fullness; prolonged gastric emptying time	Several clinical trials of varying methodological quality, mostly focused on effects on lipid and blood glucose levels, have found little to no effect on body weight	Tablet forms may cause esophageal obstructions. Other reported adverse effects include loose stools, flatulence, diarrhea, constipation, and abdominal discomfort
Green coffee bean extract (Coffea aribica, Coffea canephora, Coffea robusta)	Inhibited fat accumulation; modulated glucose metabolism	Few clinical trials of poor methodological quality have suggested possible modest effect on body weight	Reported adverse effects include headache and urinary tract infections
Green tea (Camellia sinensis) and green tea extract	Increased energy expenditure and fat oxidation; reduced lipogenesis and fat absorption	Several clinical trials of good methodological quality on green tea catechins with and without caffeine have shown possible modest effect on body weight	Reported adverse effects for green tea extract include constipation, abdominal discomfort, nausea, increased blood pressure, liver damage
Guar gum	Bulking agent in gut; delayed gastric emptying; increased feelings of satiety	Several clinical trials of good methodological quality have found no effect on body weight	Reported adverse effects include abdominal pain, flatulence, diarrhea, nausea, and cramps
Hoodia (Hoodia gordonii)	Suppressed appetite; reduced food intake	Very little published research in humans, but results from one study suggest no effect on energy intake or body weight	Concern for increased heart rate and blood pressure. Other reported adverse effects include headache, dizziness, nausea, and vomiting
Pyruvate	Increased lipolysis and energy expenditure	Few clinical trials of weak methodological quality suggest possible minimal effect on body weight and body fat	Reported adverse effects include diarrhea, gas, bloating, and possible decreases in high-density lipoprotein levels
Raspberry ketone	Altered lipid metabolism	Studied only in combination with other ingredients. Unable to draw conclusions.	Unknown
White kidney bean (Phaseolus vulgaris)	Acts as a “starch blocker, interfering with breakdown and absorption of carbohydrates	Few clinical trials, all of poor methodological quality, suggest possible modest effect on body fat, but no effect on body weight	Reported adverse effects include headache, soft stools, flatulence, and constipation
Yohimbe (Pausinystalia yohimbe, yohimbine)	Hyperadrenergic effects	Very little research has been done on yohimbe for weight-loss, with insufficient evidence to draw firm conclusions.	Significant safety concerns reported, with adverse effects including headache, anxiety, agitation, hypertension, and tachycardia
*Table adapted from The ODS Fact Sheet on Dietary Supplements for Weight Loss (294) +Listed in order of severity, with the most severe reported side effects listed last.

In 2001, MetaboLife® 356, an Ephedra-containing combination supplement, was the top selling dietary supplement. It
reached $70 million in sales, but it was also responsible for 64 percent of all herb-related adverse events reported to the U.S. Poison Control Center during that same year (175;177). Ephedra, or Ma Huang, is the common name for the herb that was used in many of these weight loss supplements. It is an herb used in traditional Chinese Medicine (TCM). Its use in weight reduction though, is not a common practice in TCM. Americans used this supplement as a weight loss aid from the mid 1990’s, up until 2004, when it was banned by the FDA (178;179).

The NIH sponsored a thorough systematic review of the safety and efficacy of Ephedra through the Agency of Healthcare Research and Quality’s (AHRQ) Evidence Based Practice Center at the University of Southern California, which conducted the study. It concluded that the use of Ephedra, with or without caffeine, correlated with a small but nonetheless statistically significant increase in weight loss over six months, (almost equal to 0.9 kilograms per month more than with the placebo). The weight lost by those taking Ephedra in combination with caffeine exceeded weight lost by prescription medications in two head-to-head randomized, double-blinded clinical trials (178). There were no studies that measured the long-term effects (more than 6 months) of Ephedra use, and the problem was that the supplement was not safe. Adverse effects of the supplement in the AHRQ study included two to three times more nausea, vomiting, psychiatric symptoms such as anxiety and change in mood, autonomic hyperactivity, and palpitations when compared with placebo. Serious adverse events (SAE’s) were defined as specified by FDA criteria. SAEs were reported to the FDA, and adverse event reports from a manufacturer of Ephedra-containing dietary supplements were also evaluated in the RAND/Southern California systemic review. These reports raised concern about the safety of dietary supplements containing Ephedra due to the number of deaths, myocardial infarctions, cerebrovascular accidents, seizures, and serious psychiatric illnesses in young adults, data was sufficient to warrant concern (178).

The FDA concluded in 2004 that Ephedra-containing products were not to be recommended for weight loss. There was unreasonable risk for illness and injury when taking such dietary supplements. Thus, the sale of dietary supplements containing Ephedra has been prohibited in the United States since April 2004 (178). Ephedra like supplements such as Citrus aurantium (Bitter orange) may also pose risk.

The latest information on dietary supplements and weight loss can be found at www.ods.nih.gov.

9.8 Fiber

The next section outlines dietary fiber needs during weight reduction.

9.8.1 Fiber Needs in Reducing Diets

Dietary fiber is chemically similar to carbohydrate in most of its forms but it is virtually non-caloric because the human body lacks the enzymes to break the fiber’s glycosidic bonds. Some short-term experimental and several cross-sectional studies suggest that an increased dietary fiber intake reduces weight gain. In contrast, fiber is not effective as a weight loss aid (180). Fiber should be included in reducing diets at levels of about 25 to 38 grams per day to facilitate laxation. Both soluble and insoluble dietary fiber may also modify hunger and help to sustain satiety, but again experimental evidence is not conclusive (10;181;182). Inclusion of five or more servings of fruits and vegetables daily, with plenty of whole grain breads and cereals helps to meet both soluble and insoluble fiber goals on reducing diets. On a VLCD, it is also important to include at least some fiber. As dietary fiber intakes increase, water requirements also increase, so intakes of fluid should also be substantial. Adequate fiber and water are essential for maintaining a soft stool and normal laxation. Ample fiber intakes are associated with reduced risk of several chronic diseases (183).

9.9 Energy-Dense Beverages, Alcohol, and Energy-Free Artificially and Naturally Sweetened Beverages

Currently, it is estimated that the mean intake of ”added” sugars in the American diet is about 15.8% of total energy, and that the largest source of these added sugars is from calorically sweetened beverages such as soft drinks, fruit aides, and other sweetened beverages, accounting for 47% of total added sugars in the diet (184;185) . The term “energy-dense beverage” encompasses a wide variety of beverages, including sugar sweetened soda, fruit drinks, juice, lemonade, sweetened iced tea, milk and soy beverages, and alcohol. Alcohol is especially calorie dense (7 Cal/gm, vs 4 for sugars), and it also may bypass satiety mechanisms and lead to a lack of ability to control eating. Curiously, in recent years it has been neglected in spite of the fact that many adults (and unfortunately some younger people) drink alcohol on a regular basis.

9.9.1 Energy-Dense Beverages

When compared to water or energy-free beverages, consumption of energy-containing beverages tends to increase total energy intake from meals (184). Although the evidence is not highly conclusive, it is argued that: 1) consumption of an isocaloric beverage compared to consumption of solid food prior to a meal increases food intake, 2) solid foods enhance satiety hormones more than energy-dense beverages, and 3) energy-dense beverages are often comprised largely of refined carbohydrates, which stimulate fewer satiety signals than unprocessed carbohydrates, fat or protein (184). More research is needed on this issue to validate each of these assumptions. It should be noted that alcohol is also energy dense and bypasses satiety mechanisms. This is discussed below further in section 9.9.2 Alcohol.

The Beverage Guidance Panel recommends that the average person limit daily consumption of caloric, sweetened beverages without nutritional benefits (soft drinks, fruit drinks, fruit cocktails, fruit aids, and sweetened teas and coffees) to eight ounces per day. Caloric, nutrient-dense beverage consumption (milk, soy and 100% fruit and vegetable juices) should be kept to a minimum (186). In the United States, a regular, 12-ounce can of calorically sweetened soda provides approximately 150 calories, typically in the form of high-fructose corn syrup. These calories, if not balanced with exercise or a caloric reduction in other areas of one’s diet, could gradually lead weight gain over time (187).

While there may be health benefits to consuming energy and nutrient dense beverages such as milk and 100% fruit juice, the additional energy provided by these energy-dense beverages must be offset by an increase in energy expenditure or a decrease in other areas of energy consumption in order for weight loss or weight maintenance goals to be achieved. Any energy-dense beverages included in the diet should also be nutrient dense, such as various forms of milks (e.g. diary, soy) and 100% fruit juices (which should be limited to about one serving per day). Most other caloric beverages, such as sodas, fruit drinks, sweetened coffees and teas provide calories with little or no vitamins, minerals, or other nutrients. For this reason, energy-dense beverages are generally not recommended for patients attempting to lose weight.

9.9.2 Alcohol

Alcohol (ethanol) contains approximately seven calories per gram, providing more energy per unit of weight than either carbohydrate or protein (each providing about four calories per gram), but less than fat (about 9 calories per gram). Alcoholic beverages are a source of non-nutritive energy, or “empty calories”. If protein, carbohydrate and/or fat are consumed at the same time as alcohol is ingested, their oxidation will be suppressed (most notably fat oxidation), since alcohol is preferentially oxidized, and the other macronutrients balance through the sparing effect of alcohol on fat oxidation. This may lead to increased fat storage.

In addition to alcohol’s influence on macronutrient metabolism, chronic extremely excess intake of alcohol also interferes with the absorption and utilization of several vitamins and minerals. Alcohol in excess also impairs nutrient absorption by damaging the stomach and intestinal lining, disabling the transport of some nutrients into the blood. Chronic overconsumption of alcohol can also lead to fatty liver, dyslipidemia, and further weight gain, and should be discouraged.

Another important consideration concerning alcohol’s influence on energy balance is its effects on energy intake. Alcohol is positioned at the bottom of the hierarchy of satiating efficiency of metabolic fuels consumed by humans (10). Generally, satiety provided by fuels is ranked from lowest to highest: alcohol, fat, carbohydrate (depending on type), and protein (188). Alcohol energy is additive to the diet, producing no compensation in energy intake under most ad-libitum situations, and in fact, some research suggests that alcohol may stimulate appetite (189;190). For these reasons, alcohol consumption is usually contraindicated on weight-loss diets.

9.9.3 Low and No Calorie Sweeteners

These ingredients are variously called low and no calorie sweeteners, non-nutritive sweeteners, sugar substitutes, reduced calorie sweeteners, or artificial sweeteners). They are added to foods and beverages to provide sweetness without adding significant amounts of sugar and calories to the product. They are also used as flavorings to mask the bitter taste of drugs http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes.

Low calorie sweeteners are appealing because they satisfy our innate preference for sweetness without the associated calories consuming sugar would have. They do not contribute to dental caries, may help to make low calorie diets more palatable, and assist compliance. These low calorie sweeteners are consumed by approximately one fifth of U.S. adults (191). In human studies, data have been conflicting on whether they are associated with weight loss (192). However, the draft conclusions of the 2015 Dietary Guidelines Scientific Advisory Committee concluded that there was moderate and generally consistent evidence from randomized clinical trials conducted in adults and children supporting the contention that replacement of sugar-containing sweeteners with low calorie sweeteners reduces calorie intake, body weight and adiposity. However evidence was judged only limited from long-term observational studies in children and adults that there was an association between low calorie sweeteners and body weight compared to sugar containing sweeteners. The Committee further concluded in its draft statement that there was only limited long-term observational study evidence in adults that there was an association between low calorie sweeteners and risk of type 2 diabetes.

The FDA has approved seven non-nutritive sweeteners for use in foods and beverages in the United States to date. Most are regulated as food additives, including acesulfame-potassium (Acesufame-K. Ace-K, Sunett, Sweet One), aspartame (Equal, NutraSweet), saccharin (Necta Sweet, Sugar Twin, Sweet ‘N Low), sucralose (Splenda), and two products that are used largely as ingredients rather than as table top sweeteners, neotame, and most recently, advantame. Cylamate is not sold in the United States but is sold in many other countries as a tabletop sweetener. The FDA granted stevia leaf, often called “sweet leaf”, a plant-derived sweetener, (PureVia, Sun Crystals, and Truvia Generally Recognized As Safe (GRAS) status. It is derived from stevia leaves by steeping them in water and purifying to extract to obtain only high purity rebaudioside A. The ingredient is sold under the trade name Rebiana and is the major source of sweetness in the Truvia sweetener brand. , Also GRAS is the product Fruit-Sweetness™ is anon-caloric fruit concentrate sweetener derived from the monk fruit, a traditional fruit originating in Southeast Asia (193). The sweeteners have a variety of different chemical structures, as shown in Table 20, and summarized elsewhere (http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes).They also vary widely in their sweetness and in their most common uses. The most commonly consumed source of these products is as a sweetener to beverages either provided by sachets of the different products, that are added to the drink or as an ingredient in a variety of “diet’ soft drinks and colas. The sweeteners used in foods and beverages are listed on the ingredient list for those who prefer a specific ingredient.

There is little evidence that low calorie sweeteners in and of themselves can cause patients to lose weight; their use must always be coupled with a hypocaloric diet. The low calorie sweeteners may make reducing diets more palatable and encourage compliance, but this remains to be demonstrated conclusively (194-195). During weight maintenance, water and other non-caloric beverages or, low calorie sweetened beverages are a preferable alternative to high calorie beverages, such as regular soft drinks, sweet teas, other sugary drinks and alcohol. Some recommend that adults consume no more than 32 ounces of low calorie sweetened beverages per day, but there is little evidence supporting such recommendations (186). If an individual wishes to reduce his/her exposure to a particular low calorie sweetener, both consuming less or choosing a variety of low calorie sweeteners are useful strategies to consider

9.9.3.1 SAFETY OF LOW CALORIE SWEETENERS

The safety of low-calorie sweeteners has been evaluated by several bodies, including the US Food and Drug Administration (FDA), the Joint Expert Committee of Food Additions (JECFA) of the United Nations Food and Agricultural Organization (FAO), the World Health Organization (WHO), the Scientific Committee on Food (SCF) of the European Commission, and the European Food Safety Authority (EFSA) (242, 254). In the United States, the use of sweeteners is regulated by FDA under the 1958 Food Additives Amendment of the Food, Drug and Cosmetic Act of 1938 (249). Currently, two low calorie nutritive sweeteners sugar alcohols (mannitol and xylitol) and six non-nutritive sweeteners (aspartame, acesulfame-K, saccharin, sucralose, neotame, advantame) are approved as food additives by the FDA (242), and two additional “natural” sweeteners, extracts of stevia and monk fruit, are approved as GRAS.

An Acceptable Daily Intake (ADI) is established for all FDA-approved low-calorie sweeteners. It represents the amount of each sweetener that can be safely ingested daily by a human over a lifetime without risk based on animal toxicity studies. The ADI is expressed as milligrams per kilogram of body weight. The ADI is not a maximum intake level. It is a conservative estimate of the level-- a hundredth of the maximum level-- at which no observed adverse effect is seen in feeding studies of toxicity in experimental animals (243). Consumption of these low-calorie sweeteners at levels below the ADI is considered by the FDA to be safe for the entire population, including children and pregnant women (246). Exceptions for particularly vulnerable populations, such as individuals who suffer from phenylketonuria (a rare metallic disorder), who should not consume aspartame-sweetened products, are listed in the table, and on the product label.

Low-calorie sweeteners such as stevia and monk fruit extracts that are GRAS are also are acceptable only for specified uses in specified amounts that are in line with traditional uses of these ingredients in human diets over the course of history. Generally Recognized as Safe (GRAS); substances are those that scientific experts have agreed are safe for use in appropriate amounts in foods and beverages, based largely on traditional use. They do not require the extensive testing required by FDA for food additives, but are permitted to be used only at specified levels (244). Also, some other compounds with sweet tastes, including thaumatin, neohesperidine, and glycyrrhizin are GRAS when used in small amounts for an intended use as flavor enhancers, but not as sweeteners.

9.9.3.2. Low calorie sweeteners that are currently approved by FDA:

Saccharin—Sold under the brand names Sweet ‘N Low®, Sugar Twin®, and Sweet Twin®, saccharin is a non-nutritive sweetener that is not metabolized by the body and provides no calories. It has been on the market longer than any other low calorie sweetener. It is 300-500 times sweeter than sugar and is heat stable (246), but leaves a bitter metallic aftertaste in the mouth in individuals who have a certain dominant genetic trait. They can be identified by the ability to taste the chemicals ptc (phenylthiocarbamide) and PROP (6 propyl 2 thiouracil) as bitter or metallic. Those compounds do not appear in food but related compounds such as saccharine and possibly some of the Brassica vegetables have related compounds that are also tasted as bitter. About 70 % of Americans taste ptc related compounds in food as bitter, especially if they are not habituated to coffee or tea or non-smokers, and therefore in the USA saccharine is usually blended with other sweeteners in food and beverage products (252). Many Asians are very sensitive to a bitter taste, and formulations of low calorie soft drinks for them may use other sweeteners (247). The ADI for saccharin is 15 mg/kg/day. Studies in the 1970s in rats linked a lifetime exposure to high doses of saccharin to development of bladder cancer. This raised concerns about the safety of saccharin and led to a ban of saccharin in 1977 by the FDA, and great furor since at the time there were few other non-nutritive sweeteners on the market. Later, after the ban was lifted due to Congressional action, FDA required that products containing saccharin must carry a warning label (244). Subsequent human case- control and other studies showed no association between saccharin and bladder cancer development (254). In addition, it was later shown that physiological differences in bladder and urine chemistry between rats and humans were such that the bladder cancer-causing effect was specific only to rats, and that the toxic effect of tumors in rats was evident only with sodium saccharin but not with other forms of saccharin, and only with lifetime exposures to very high dosages of saccharin, equivalent to hundreds of servings per day in humans (247). After these studies were reviewed, in 2000 saccharin was delisted as a potential human carcinogen. However, products containing saccharin must still label saccharin in declarations on their ingredient list. Today, several professional societies, including the American Dietetic Association, American Medical Association, and American Cancer Society state that saccharin is safe and acceptable for use in all populations (242).

Acesulfame K (Ace-K)—Under the brand names of Sunett® and SweetOne®, Ace-K is a combination of an organic acid and potassium. It is excreted from the body unchanged, and therefore does not yield either a net increase in calories or of potassium in humans. It is 200 times sweeter than sugar. It is heat stable, and thus is suitable for cooking and baking (242). Ace-K also leaves a metallic aftertaste in some people’s mouths when it is used by itself, and so it is usually combined with other sweeteners in sweetener blends, especially in carbonated beverages. The ADI for Ace-K is 15 mg/kg/day; for a person of 70 kg that would be about 1050 mg. The typical amount of Ace-K in a 12-oz beverage is 40 mg and in a packet of tabletop sweetener it is 50 mg (257). Long-term human studies found no effects on cancer even at very high consumption levels of Ace-K. There are also no case reports documenting adverse health effects associated with Ace-K. Therefore, FDA permits Ace-K use in all population segments. An Ace-K metabolite, acetoacetamide, is toxic when consumed at very high doses, but the amount of acetoacetamide found in beverages sweetened with Ace-K is negligible. And therefore consumption of beverages sweetened with Ace-K is deemed to be safe (247).

Sucralose—Sucralose is sold under the trade name of Splenda®. It is a sugar derivative, which replaces 3 hydrogen-oxygen groups on the sugar with 3 chlorine atoms. Although sucralose is derived from sugar, it is not absorbed nor do gut enzymes in humans metabolize sucralose as a carbohydrate. It is non-caloric (251). It is 600 times sweeter than sugar and is highly heat stable. Sucralose is used extensively in foods and beverages because it retains sweetness over a wide range of temperatures and storage conditions (242). The ADI for sucralose is 5 mg/kg/day. Sucralose had no significant effect on blood glucose control in individuals with type 2 diabetes mellitus even at levels 3 times this amount over three months. Extensive research in humans and experimental animals has found no association between sucralose consumption and carcinogenicity or other health concerns (244). Therefore, sucralose appears to be safe and acceptable for human consumption.

Aspartame—It is sold under the trade names NutraSweet®, Canderel®, Sanecta®, TriSweet®, E951 (for food ingredients), and Equal® (for the tabletop sweetener). Aspartame is a synthetic sweetener composed of aspartic acid and phenylalanine (256). It is used both as an ingredient and sold to consumers as a tabletop sweetener. Since it is metabolized to amino acids, aspartame provides 4 calories per gram. It is 160-220 times sweeter than sugar. Due to its intense sweetness, only a tiny amount (e.g. a few mg) is needed to sweeten a food and thus, the energy provided from the compounds is negligible. Aspartame decomposes and loses its sweetness with heat and so it is not suitable for baking and cooking. However, it is used extensively in soft drinks, which account for more than 70% of aspartame consumption in the US (242). An 8-oz diet Coca Cola contains approximately 125 mg of aspartame and 60 mg of phenylalanine (257). The ADI of aspartame is 50 mg/kg/day (or about 3500 mg for a 70 kg individual). Due to the presence of phenylalanine in aspartame, individuals with phenylketonuria (PKU), a recessive inborn error of metabolism should be cautioned about aspartame consumption since they cannot metabolize phenylalanine effectively and the amino acid can accumulate in the blood, causing toxic metabolites and potentially neurological damage (242). Although this adverse effect is unlikely because the amounts of aspartame that are used in foods and as a sweetener is quite small, diet therapy for PKU includes limiting dietary phenylalanine, including aspartame. FDA requires products containing aspartame to carry the warning label “PHENYLKETOURICS: CONTAINS PHENYLALANINE.” The plasma response of phenylalanine to ingestion of aspartame varies in people with PKU, but most seem to tolerate the amount of phenylalanine in a single diet soda sweetened with aspartame (about 104 mg phenylalanine/12-oz can), so an immediate health crisis is unlikely should a person mistakenly drink a diet soda (244). However, caution is still indicated for PKU patients. In contrast, healthy adults show no significant change in plasma level of aspartic acid even with doses of aspartame about 4 times the ADI (4 times ADI is equal to 200 mg/kg/day) (256). In normal humans, plasma phenylalanine increases in a dose-response manner to aspartame dosage in the range of 2-100 mg/kg/day without any observed effects on cognitive function, but individuals with phenylketonuria are different and may be particularly sensitive (258).

Aspartame is perhaps the most controversial sweetener of them all due to early claims in the 1970S of neurotoxicity in primates, which were later not confirmed, although the unproven claims continue to circulate on the Internet. One comprehensive review study on the safety of low-calorie sweeteners, including aspartame, stated that “much of potential misinformation about aspartame and health seems to be based on misunderstandings or partial scientific truths” (247) some consumers report adverse reactions to aspartame, including headache, facial edema, skin reactions, respiratory problems, seizures, and behavioral and cognitive changes, numerous controlled studies have failed to reproduce these adverse reactions reported. Adding to the controversy, aspartame was claimed by one Italian laboratory to be carcinogenic, but no associations between aspartame consumption and cancer development were found in a review of their data by a panel of experts in the experimental studies in rodents upon which the claim was based, and the studies themselves were criticized as being inadequate (256) Other data reviewed also were negative for adverse effects. The National Cancer Institute at NIH concluded that there was a lack of association between aspartame consumption and increased cancer risk, even at high intake levels after renewing all of the data (259). The European Food Safety Authority (EFSA) panel in 2009 again reviewed all available scientific evidence on the safety of aspartame and concluded that there was no carcinogenic effect nor association with neurobehavioral disorders or other effects from it and that no further revision of the ADI was needed. The most recent statement of EFSA released in 2013 has also confirmed the safety of aspartame (260). Periodic updates will be made available. Current evidence suggests that aspartame is safe at levels below ADI other than for individuals with PKU, and there is no credible evidence showing carcinogenicity, neurotoxicity, and other adverse health outcomes even at levels above ADI.

Neotame—Neotame is a relatively new non-nutritive low calorie sweetener, approved by FDA in 2002. It is used as an ingredient and also as a flavor enhancer that can modify other flavors in foods and beverages (246). Neotame is chemically related to aspartame, being composed of aspartic acid, methanol, and phenylalanine. Although they are similar in chemical structure, neotame and aspartame are completely different compounds with different physical and biological properties (247). Neotame is 7000-13000 times sweeter than sugar, thus only an infinitesimal amount is required for sweetening. The ADI for neotame is 2 mg/kg/day in other countries and 18 mg/day in the United States. Although neotame is metabolized into phenylalanine, a PKU warning label is not required for products containing neotame because the amount of neotame to sweeten a food is so tiny, owing to its intense sweetness. Thus, exposure to phenylalanine from neotame is negligible and clinically insignificant (244). In addition, neotame is not directly metabolized to phenylalanine (252). The blockage of peptidases that break the peptide bond between aspartic acid and phenylalanine decreases the availability of phenylalanine in the bloodstream after neotame ingestion. Some concerns have been raised regarding neotame being neurotoxic due to the structural similarity to aspartame. However, no adverse effects of this sort or of other concerns have been found (258). Therefore, neotame is considered safe and acceptable for use in all populations.

Advantame This ingredient is an intense low calorie non-nutritive sweetener approved in 2014. It is used chiefly as a food ingredient, and is not sold over the counter. It was approved in 2014 for use in foods and beverages; USDA must approve uses in meat and poultry as well. It is structurally somewhat similar to aspartame (295).

Stevia (Stevioside, Steviol glycosides, Rebaudioside A)—These are sold under the trade names Truvia®, Sun Crystals®, PureVia®, Sweetleaf Sweetener®, Stevia in the Raw®, and Enliten®. Stevia leaves, often called “sweet leaf”, have been used for centuries in Asian and South American countries as non-caloric sweetener and traditional medicine (261). Steviol glycoside is the sweet component derived form leaves of Stevia rebaudiana. It is 250-300 times sweeter than sugar. Stevioside extracts have been used for sweetening in pickled vegetables, seafoods, soy sauce, soft drinks, and confectionary (262). Stevia leaves have also been used medicinally in Asia and South Africa for hypertension, hyperglycemia, obesity, and skin disorders, although the evidence for beneficial effects is scanty and none of these therapeutic uses are approved in the US. Stevioside is not metabolized nor absorbed by the digestive tract. The use of steviosides as a food ingredient was not initially approved in the United States although it was in Europe. Rather, it was sold in the US initially as a dietary supplement and therefore it could not be marketed as a sweetener since this was a different intended use. In 2008, FDA granted stevia GRAS status for use as a general-purpose sweetener in addition to use as a dietary supplement (263). Stevioside can now be found as an ingredient in beverages and as tabletop sweeteners. The Joint Expert Committee on Food Additives (JECFA) conducted a thorough scientific review on stevioside and concluded that it was safe for use in food and beverages with no major toxicity risk. The ADI of 0-4 mg/kg/day has recently been established by JECFA (296).

9.9.3.2 Nutritive low calorie sweeteners

Polyols (Sugar alcohols)—Sugar alcohols (polyols) are considered nutritive sweeteners because they provide some calories in the diet. However, the amount of calories provided per gram is fewer than table sugar (about 2 vs 4 calories/gram) due to the alcohol’s incomplete digestion and absorption. The unabsorbed polyols reach the colon and cause subsequent fermentative degradation by intestinal bacteria (297, 248). Most polyols are about half as sweet as sugar. They replace sugar in the products for sweetness and volume, and therefore, products containing them can be labeled as “sugar-free”. Due to the osmotic effect of unabsorbed polyols in the large intestine, products containing polyols may cause bloating, gas, discomfort, and diarrhea when consumed in excess (e.g. > 50 g/day sorbitol and >20 g/day mannitol). Therefore, these products are required to carry the statement “Excess consumption may have a laxative effect” on the label. Sensitivities to this laxative effect differ among individuals. Children may be particularly sensitive to the laxative effects of polyols due to their smaller body size (244). Tolerance to polyols can be increased somewhat with a gradual increase of intake that allows for adaptation to their laxative effects (264). Polyols may also confer potential health benefits, such as reduction in dental caries, a lower glycemic response when they are used in place of sugar, provision of fewer calories than sugar, and possibly a prebiotic effect (265). The prebiotic effects are presently poorly documented.

There are three subtypes or categories of Polyols

Polyolnosaccharides:

1) Erythritol—Marketed under the brand name Zerose, erythritol is found naturally in pears, melons, grapes, and mushrooms. Due to its almost complete absorption and unchanged excretion in urine within 24 hours, erythritol may not have as potent a laxative effect as some of the other polyols. In addition, erythritol provides fewer calories per gram (0.2 calories per gram) than the other polyols (253, 264).

Sorbitol—Usually found in sugar-free candies, chewing gums, baked goods, frozen desserts, and toothpaste, sorbitol is non-cariogenic. It is also slowly absorbed in the gut and metabolized independently of insulin (248, 253). Thus, it may be beneficial for individuals with diabetes.

Mannitol—Extracted from seaweed, mannitol is poorly absorbed and may cause a stronger laxative effect at a relatively lower dose (10-20 gram) than other polyols (252, 253). It is used as a dusting powder for chewing gum, an ingredient in chocolate-flavored coating agents for ice cream and candies, and in pharmaceuticals. It also cools the mouth and masks bitter tastes.

Xylitol—Xylitol is extracted from birch, raspberry, plum, and corn. It is often found in sugar-free gums, cough drops, mints, and oral health products. Several clinical trials have shown that xylitol is more effective in reducing dental caries than any of the other polyols. In addition, xylitol may even have a caries-preventive and anti-cariogenic effect (4). Due to this, FDA authorizes the health claim that “xylitol does not promote tooth decay” on product labels (256).

2) Polyol Disaccharides:
Maltitol-- Derived from maltose, maltitol is a food ingredient that is metabolized into glucose and sorbitol by the gut flora. It is used in chocolate candies, jams, baked goods, and ice cream to give texture to products, and sometimes as a fat replacer (http://www.caloriecontrol.org/sweeteners-and-lite/polyols).

Lactitol –Although it is derived from lactose, lactitol is not hydrolyzed by lactase in small intestine. Instead, it is fermented by the microflora in the large intestine and converted into biomass and short-chain fatty acids. Also, it acts as a prebiotic, stimulating growth of the gut bacteria. The health effects of this are uncertain, but because lactitol is fermented in the colon, it may act as prebiotic and may produce more intestinal discomfort than some of the other products (249, 253). Lactitol is used as an ingredient in bakery products, hard and soft candies, frozen dairy desserts, and chocolate (248)

Isomalt—Under the trade name Palatinit, isomalt is incompletely metabolized into mannitol, sorbitol, and glucose in small intestine. It is noncariogenic and triggers only a low glycemic response (242). It is approved in most European countries and is added to candies, toffee, fudge, wafers, and cough drops. 90% of the isomalt is fermented further in colon, making it also a potential prebiotic (265). Intakes of 30 grams of isomalt might increase numbers of bifidobacteria in the colon, but evidence is still uncertain on this, and even if it did the effects of increased bifidobacteria on health are not yet well documented.

3) Polyol Polysaccharides:

Hydrogenated starch hydrolysate (HSH)—HSH is produced by the hydrolysis of corn, wheat, or potato starch. Lycasin®, Hystar®, Stabilite®, and Roquette's 75/400 are the HSH brands currently available on the market. They are used mostly as bulking agents and food ingredients to provide sweetness, volume, and texture in commercially produced “sugarless” food products. HSH is metabolized into maltitol, sorbitol, and glucose to provide 3 calories per gram (253). The glycemic index of HSH is similar to that of maltitol, and about 40% of it is digested in the intestine (264). Therefore, it may be suitable for individuals with diabetes but this claim needs more evidence to support it.

9.9.3.4 Other sweeteners pending approval:

These sweeteners are not yet fully approved by FDA for use in food:

Cyclamate—Sold under the trade names SugarTwin® and Sucaryl®, cyclamate is 30 times sweeter than sugar, provides no calories, and blends well with other sweeteners. Most individuals do not metabolize cyclamate (252). It is approved for use as a tabletop sweetener in more than 50 countries, but re-approval is currently held abeyance and pending in the United States. Cyclamate has been banned in the US since 1969 based on a study suggesting the association between a saccharin/cyclamate blend sweetener and bladder cancer in rats (266). Cyclamate is converted to its metabolite, cyclohexylamine, which is relatively toxic and may cause bladder cancer in rats. High doses of cyclohexylamine cause male infertility in rats, although this effect is not observed in humans (247). Studies since 1969 have found no relationship between cyclamate and bladder cancer in humans, and this is the evidence that is currently being considered by FDA pending re-approval. FDA has stated that animal studies on mice and rats do not implicate cyclamate as a carcinogen.

Alitame – Under brand name Aclame®, alitame is derived from amino acids alanine and aspartic acid. Only the aspartic acid component is metabolized, to yield 1.4 calories per gram. Since only a minute amount of the sweetener is used as a food ingredient, the energy provided is negligible. It is 2000 times sweeter than sugar and heat stable. Common applications of alitame are commercially produced baked goods, candies, frozen desserts, beverages, and pasteurized foods. Alitame has been approved by several countries and is pending approval in the USA (242, 253).

Thaumatin—Sold under trade name Talin®, thaumatin is a protein sweetener that occurs naturally in a West African fruit Thaumatococcus danielli. It is 1600 times sweeter than sugar. However, it leaves a licorice-like aftertaste. Its application in food products is very limited due to the delayed appearance and extinction time of the sweet sensation and this licorice aftertaste (249). In the United States, thaumatin is recognized as GRAS as a flavor enhancer (especially in chewing gums), but not as a sweetener. The Joint Expert Committee on Food Additives (JECFA) reviewed the safety of thaumatin and found no toxicity. The ADI for thaumatin is not yet specified (244).

Neohesperidine dihydrochalcone – A derivative of bioflavonoids in citrus fruits, neohesperidin dihydrochalcone is 1500 times sweeter than sugar but has different flavor profile. It also leaves a licorice aftertaste. However, it intensifies the mouth feel of juices, and thus has the potential to be used in fruit juice, chewing gums, and mouthwash (242). It is metabolized by the gut flora. Although it is approved in the European countries, it is recognized as GRAS only in amounts for use as a flavor enhancer in the United States, but not for the larger amounts that would be needed for use as a sweetener

Glycyrrhizin—An extract from licorice root, glycyrrhizin is 30 times sweeter than sugar (247). It has limited use as a flavoring agent in some candies and tobacco due to its strong licorice flavor. It is also GRAS in the United States as a flavoring agent, flavor enhancer, and surfactant, but not as a sweetener (249).

9.9.3.5 POSSIBLE HEALTH BENEFITS OF LOW CALORIE SWEETENERS:

Weight loss and maintenance

At different times and places overindulgence of various foods has occurred. In the US today many people of all ages indulge in sugary sweet foods and beverages high in calories, and adults also in alcohol. Reduction of food energy from any source, including these sources, will result in weight loss over the long term if other foods are not substituted for it. In 2014 the Obesity Society issued a statement that reduced consumption of sugar-sweetened beverages can reduce total caloric intake and that individuals and especially children and those with weight problems reduce their consumption of sugar sweetened beverages (298). Low calorie sweeteners offer an option other than water for alternative beverages, but the reality remains that in order to lose weight, the dieter must achieve and maintain a negative energy balance (e.g. Energy intake < energy expenditure) without compensating for what is e decreased by increasing intakes of other foods or beverages. . Some randomized-controlled studies have shown beneficial effects of low calorie sweeteners in weight management; with a small short-term weight loss (of about 0.2 kg/week) over placebo with the low calorie sweeteners plus a hypocaloric diet, and improved weight maintenance after the weight loss diet is discontinued while others do not (242, 267, 268, 269, 270). Another study found that when the artificial sweetener group (particularly aspartame) was compared to the control it showed a difference of about 5 kg (maintained a weight loss of 5.1 kg longer), when artificial sweeteners were substituted for caloric sweeteners in beverages and with a hypocaloric diet (267). Other studies were not as positive. A review of aspartame’s role on weight control in 2006 showed a weight loss of 0.2 kg/week when products sweetened with sucrose was substituted with aspartame (269) in a hypocaloric diet. Another study showed more successful maintenance of weight loss after three years in women who were encouraged to consume aspartame-sweetened products (247).

In the most definitive study to date, of 303 participants who were overweight, during a 12 week weight loss program those who used low calorie sweetened beverages instead of water alone lost more weight than those in the control group (5.95kg vs 4.09 kg) and felt less hungry as well (299). However, in all of these studies, a weight loss regimen was essential as well, and without one it is likely that compensation would occur, wiping out any beneficial effects. The 2004 position statement of the American Dietetic Association therefore concluded that, “nonnutritive sweeteners have the potential to promote weight loss in overweight and obese individuals”. Low-calorie sweeteners provide sweetness and palatability to food, with only minimal or virtually no food energy calories. Substituting full-calorie products with low-calorie sweetened product results in fewer calories consumed (244). Assuming that there is no eventual calorie compensation in the regulation of food intake that should be helpful, but such compensation may in fact occur over time without a conscious effort on the dieter’s part to reduce intakes especially when people know they are consuming less calories. Calorie reduction may be achieved only when and as long as low-calorie sweeteners replace their full-calorie counterpart without any additional food eaten to compensate for amount of calories saved.

Because excessive sugar intake might contribute to weight gain and obesity, owing to its effects on increasing caloric intake, a reduction in sugar intake through substitution of low-calorie sweeteners has been proposed to help prevent weight gain. The “AmericanOnTheMove” study in 2007 was conducted to investigate the effect of changes in diet and physical activity on excessive weight gain in overweight children. It showed that a replacement of dietary sugar with a low calorie sweetener (sucralose) in the diet might be an effective tool to reduce caloric intake and reach negative energy balance. However, it should be noted that the experimental group in the study was provided with Splenda products and compensation for participation in the study and these may have been added incentives to patients (300). Thus additional work is needed before the finding can be judged to be well established.

The notion that low calorie sweeteners increase hunger or cravings by uncoupling the sensory and hedonic aspects of sweetness from their satiating effects and thus leading to overconsumption of sugary foods has not been borne out in recent studies. The appetite for sweetness does not seem to be increased by their use and there is no increase in appetite or hunger with consumption of low-calorie sweetened foods and beverages. (301)
(242, 272). One American Dietetic Association’s position statement on nonnutritive sweeteners concluded that nonnutritive sweeteners had no effect on appetite, hunger, and fullness in adults at least, based on results from short-term studies (244). A more recent statement of the Academy of Nutrition and Dietetics in 2012 found that the low calorie sweeteners when substituted for nutritive sweeteners might help consumers to limit carbohydrate and energy intakes as a strategy to manage blood glucose or weight. The American Heart Association/ American Diabetes Association stated in 2012 that some data suggested that low calorie sweeteners might be used in a structured diet to replace sources of added sugars, and that this substitution might result in modest energy intake reductions and weight loss.

Low calorie sweeteners are also claimed to increase adherence in weight-loss programs by providing more palatable food choices from low-calorie sweeteners, and increasing satisfaction with the weight-loss diet (243, 246). These findings also need to be replicated, but it does appear that they have a neutral or slightly positive effect when accompanied by a hypocaloric diet.

Improved quality of the diet

When the caloric intake from added sugar exceeds 25% in usual diets, the amount of some micronutrients in the diet may be reduced and diet quality worsened (244). The theory is that by replacing foods and beverages containing added sugar with the ones containing low-calorie sweeteners, calories can be saved for consumption of more nutrient-dense foods and diet quality may be improved. For example, such products might be helpful for elderly who have decreased energy needs and people who are sedentary and thus require lower caloric intake. However, there is no definitive study to date that explores whether the relationship between use of low-calorie sweeteners and diet quality does in fact exist. One study compared nutrient and energy intakes, quantity of food consumed, and the knowledge and practices between the reduced-sugar foods users and the users of the full sugar versions of the same products (273). The study found that users of reduced-sugar products had higher micronutrient intakes as they reported higher fruits, lower intakes of added fat and sugar, and more label reading. However, this study was cross-sectional and only provided information at one point in time but not over time. Better-designed studies are needed to evaluate relationships between the use of low-calorie products and diet quality.

Diabetes management

Type 2 diabetes is common and appears to be on the rise. The cornerstone of dietary therapy remains weight reduction if the patient is obese, and selected medical options including orlistat (Xenical), phentermine/lopiramate (Quymia) and lorcaserin (Belviq) to assist weight loss and various other medications to assist in the lowering and control of blood glucose levels. Intensive lifestyle interventions that promote weight loss in patients with type 2 diabetes have better outcomes than standard diabetes support and education, including use of fewer medications, lower health care costs and fewer hospitalizations (302). Since low-calorie sweeteners contain virtually no calories and no carbohydrates, they may help people with diabetes to use less sugar and make more healthful food choices, especially if they are trying to lose weight and control their blood sugar levels. However, weight loss will not occur if the dieter substitutes other foods for those that have been eliminated. The American Diabetes Association recommends the use of artificial sweeteners in a calorie controlled diet that also controls carbohydrate intake as part of medical nutrition therapy for diabetes, to better maintain blood glucose levels near normal and control carbohydrate intake (274).

The European Food Safety Authority (EFSA) reviewed data to substantiate health claims related to ability of low-calorie sweetener to reduce post-prandial glycemic response and the use of low-calorie sweetener and glycemic response in people with impaired glucose tolerance. It concluded that such a claim was valid, but that there was no causal relationship between replacement of sugar-sweetened foods and beverages with low-calorie sweeteners and maintenance of normal blood glucose levels (275).
The Glycemic Index (GI) is defined as “the incremental area under the blood glucose response curve of 50 grams carbohydrate portion of a test food expressed as a percentage from a standard food taken by the same subject” (265). Foods with low GI value produce only small rise in blood glucose after ingestion, and thus may aid in control of blood sugar. All sugar alcohols are categorized as low-GI foods. In addition, certain sugar alcohols, sorbitol and xylitol, cause slow increase in plasma glucose due to delayed absorption and metabolism in the liver as well as providing low glycemic responses. Therefore, they may be helpful tools for managing blood glucose levels for people with diabetes. A recent systematic review on Stevia proposed the use of stevia (stevioside) as an antihyperglycemic agent. Stevioside may decrease glucose levels by stimulating the production of insulin, based on studies in humans and animals (262). Another recent study on the effects of stevia, aspartame, and sucrose on postprandial glucose and insulin levels found reduction in postprandial blood glucose levels with consumption of stevia before lunch and dinner meals when compared to both aspartame and sucrose (272). However, better-designed studies on humans are needed to determine the effect of stevioside on glucose tolerance and insulinemia. A recent report hypothesized that low calorie sweeteners increased the risk of altered glucose tolerance and diabetes by altering the microbiome in the gut (Suez et al doi:1036/naturea13794). The claim was based on studies in mice fed very large doses of saccharin, aspartame or sucralose sweeteners which the workers claimed altered insulin resistance. However, when one low calorie sweetener, saccharin, was fed to 7 volunteers, blood sugar rose in 4 and did not change or diminished in the other 3. Other studies with larger sample sizes and more rigorous designs have failed to show adverse effects on glucose tolerance (303)

Reduction of Dental Caries

Dental caries is a complex disease, also involving teeth, bacteria, sugar, time, and saliva. Dietary sugar intake increases the risk of developing dental caries. Sugar (especially retentive forms of sugary foods that stay in the mouth) is metabolized by mutans streptococcus and other cariogenic bacteria on the surfaces of unclean teeth into acids, reducing the pH of the enamel and eroding it, causing decay. When the pH level falls below 5.5, tooth enamel demineralizes through the loss of calcium and phosphate ions (245). Demineralization can be reversed at the stage before the cavity is formed. Decreased sugar intake and good dental hygiene are an important strategy for prevention of dental plaque and dental caries. Therefore, substitution of low-calorie sweeteners (which are not fermented by the bacteria in the mouth) for sugar reduces sugar content of the diet, and thus may help prevent the development of dental caries (276). Sugarless gums and sugarless cough drops, mints, and lozenges also are non-cariogenic, which will not cause dental caries.

It is well known that the low calorie nutritive sweeteners, the sugar alcohols, especially xylitol, have anti-cariogenic properties and prevent formation of dental caries. Polyols are generally not substrates for bacteria in dental plaque. They are not metabolized by the bacteria in dental plaque into acids. The lack of acid production prevents against demineralization of teeth and subsequent dental caries formation (245, 265). However, when polyols (especially sorbitol), are consumed in large amounts (for example, more than two sticks of chewing gum in one day) in animal studies, in one animal study, the number of sorbitol-fermenting bacteria in the mouth increased slightly, somewhat reducing this anti-cariogenic effect. Nevertheless, the fermentation of sorbitol remained very slow when compared to that of sucrose (277). Thus, the bacteria in the mouth adapt to restriction of sugar by alternatively metabolizing sorbitol, but the net effect of the substance still remains positive. More research is needed to on these effects in humans.

Plaque formation may also be affected. In addition to their non-carcinogenicity, polyols also slow demineralization of tooth and promote remineralization of teeth that are with early lesion in the demineralization process. Chewing sugar-free gums containing polyols stimulates more saliva flow in the mouth. Saliva, which has high pH, acts as a buffer to acid production and washes away sugar and acids that could accumulate bacteria. In addition, saliva provides calcium and phosphate ions that can remineralize tooth enamel in the initial stage of demineralization before formation of carious lesion (265, 277).

Among all the polyols, xylitol is the most effective in reducing and preventing dental plaque and caries. Xylitol is the least fermentable polyol. Cariogenic bacteria do not ferment it and thus, the pH of dental plaque does not decrease and the enamel is not eroded (245). Xylitol also inhibits the growth of plaque-forming bacteria themselves (particularly the mutans streptococci) because it is a poor substrate for the bacteria. Therefore, can reduce the accumulation of plaque on tooth enamel (277). The structure of xylitol allows it to form a complex with calcium ions (in a chelate-like structure), which can promote remineralization of tooth enamel, resulting in reversal of the early dental caries lesion (250). Xylitol also acts as a bacteriostatic agent, through the conversion of xylitol into xylitol-5-phosphate by some strains of streptococci. Xylitol-6-phosphate degrades cell membrane of the bacteria and thus, reduces plaque quantity and adhesivity (265). Interestingly, xylitol can also help prevent intra-familial transmission of mutans streptococci from mothers to infants through its property to reduce mutans streptococci quantity (250, 277). Infants can be infected with mutans streptococci through oral transmission from mothers. Infants whose mothers chewed xylitol-sweetened gum had lower counts of mutans streptococci. Therefore, xylitol may also help protect against maternal transmission of cariogenic bacteria.

9.9.3.6 POSSIBLE HEALTH CONCERNS:

Safety

In the USA the Food and Drug Administration permits the sale of all of the low calorie sweeteners mentioned above. In the European Union, cyclamate is also allowed and considered safe.

The products have been judged to be safe for consumption by pregnant women. However, one low calorie sweetener, aspartame (sold as NutraSweet and Equal), should be avoided in pregnancy by women who are homozygous carriers of phenylketonuria because it is metabolized to phenylalanine. In such women at very high levels phenylalanine crosses into fetal circulation and may increase risks of mental retardation. In pregnant women, there is one report that more than one diet drink per day increased risk of preterm delivery (OR 1.38, 1.15-1.65) but this has not been confirmed (304) However, other investigators have not been able to confirm the finding. The European Food Safety Authority recently conducted a full risk assessment on aspartame in view of concerns on the part of consumers that included both animal and human studies. Its’ conclusion, issued in 2013, was that aspartame and its breakdown products were safe for human consumption at current levels of exposure (the ADI for aspartame is 40 mg/kg/day). It added that for patients with phenylketonuria, the ADI was not applicable because they require strict adherence to a diet low in phenylalanine. However threw a no risk to developing fetuses at the current ADI with the exception of women suffering from phenylketonuria. (296). The Dietary Guidelines 2015 Advisory Committee’s draft conclusion for its final report concurred with the EFSA Panel and judged the evidence as moderate that aspartame in amounts commonly consumed was safe and posed minimal health risk for healthy individuals without PKU (including those at risk of most cancers, seizures and cognitive/behavioral problems in children and adults) (305).

Other risks are not well documented. For example, in some observational studies, low calorie sweeteners have been found to be associated with risks of metabolic syndrome (306, 307), with type 2 diabetes (308), or with coronary heart disease and kidney disease (309). However, other observational data show that there is an absence of adverse effects. For example, in the CARDIA study, those consuming a healthy diet pattern with diet beverages showed lower risks of elevated glucose and low HDL cholesterol than did others (310). More impressively in a randomized clinical trial, the CHOICE study, it was found that the diet drinks group showed declines in blood pressure and fasting glucose that were no different than those of controls. The goal was to replace caloric beverages with water or diet beverages to assist weight loss in this randomized single center single blind study of 318 overweight or obese adults studied over a 6 month period. The subject received water alone plus a monthly group website class, a low calories sweetened diet beverage along with the monthly group website, or only the website education on weight loss. None of the groups achieved large weight losses; the largest was the diet beverage group with a loss of 2.5% of body weight, and the smallest was the attention control with 1.5% weight loss at 6 months. The group consuming the diet beverage showed a greater likelihood of achieving a 5% weight loss compared to the attention control (OR 2.29, 95% CI 1.05, 5.01 P=0.04) while the water group did not differ from the control in terms of its likelihood of achieving a 5% weight loss (311, 312). Effects were similar in another study that included weight maintenance. Dieters were given added advice to use the low calorie sweetener aspartame in a yearlong multidisciplinary program of weight loss and two year follow up, while others were not. The differences between the two groups were small but during maintenance the low calorie sweetener users kept more weight off than those who were non-users (267).

Even among those who use many low calorie sweetened foods and beverages, there is little likelihood that they will reach the ADI. For example, one would have to eat 75 low calorie sweetened yoghurts a day for one’s entire lifetime to achieve the ADI for one common low calorie sweetener. In children there is a concern that low calorie sweetened beverages might displace milk and 100% juice, although this fear is not well documented by evidence it has led to regulations that do not include low calorie sweeteners as permitted foods in elementary schools. The American Academy of Pediatrics has recommended that nonnutritive sweeteners should not form a “significant part “ of a child’s diet, and the Academy of Nutrition and Dietetics views them as safe for children within the range of the ADI (244).

Concern has been expressed based on some observational studies, that low calorie sweetened beverages might be associated with long-term weight gain (313, 314), but others show decreased body weight (315), or both increases and decreases (316). However, in a recent met-analysis of 15 randomized controlled trials, which are considered to be superior in assessing causal inference, as well as 9 prospective cohort studies, the correlation between low calorie sweeteners and body weight favored them very clearly over the comparator arms in the randomized studies, and were not significant in the prospective cohort studies (317). The results suggest that other factors, including reverse causation (that is, low calorie sweetener users being more likely to be obese, gaining weight and trying to control their weight gain rather than the sweeteners causing the weight gain may have been present. People who have maintained their weight loss over the long term use fewer sugar sweetened beverages and more low calorie sweetened beverages than to always normal weight persons (318), and in the US National Weight Control Registry long term weight maintainers also used more water, low calorie sweetened beverages and less alcohol and sugary drinks and juices.

Increased appetite and intake

There have been claims of a paradoxical effect of low-calorie sweeteners on appetite stimulation, leading to an increase in food intake. A 1986 study from the United Kingdom (278) compared the effect of water sweetened with aspartame versus plain water on hunger level. Those consuming highly sweetened water rated their hunger level higher than those who consumed only water. However, the study examined only perceived hunger level, which was subjective, and did not really assess participants’ actual food intake. A subsequent study showed no increase in actual food intakes 1 hour after consumption of solutions sweetened with saccharin, aspartame, and acesulfame-K (279).
Another theory of how low-calorie sweeteners might be orexigenic (enhance intake) involves stimulation of food reward pathways in the hypothalamus after ingestion of food (in this case, glucose). Sweetness stimulates the mesolimbic dopaminergic system, producing a feeling of satisfaction and can stimulate food intake (280, 281). Some evidence suggests that such pathway was not observed with ingestion of low-calorie sweeteners. One study using functional magnetic resonance imaging showed longer suppression of this signal in the hypothalamus with glucose ingestion in normal weight men, but found no such effect with sucralose ingestion. Thus, the lack of responsiveness to this signal from consumption of low-calorie sweeteners might theoretically lead to motivation to seek more food (281). However, at present, the evidence is insufficient to conclude that low-calorie sweeteners enhance appetite or food intake, and more research is needed.

Psychobiological signaling between food and the gut as well as energy compensation of meals are two other proposed explanations of how low-calorie sweeteners may lead to overconsumption and weight gain. The psychobiological theory is that low-calorie sweeteners, unlike caloric sweetener, only provide sweetness but not the food energy (282). When sweetness is not accompanied by calories, there is no signal to provoke cephalic phase of digestion in the gut to prepare for arrival of nutrients and to begin the process of energy utilization and thermogenesis. The animal model showed less effective energy regulation through this mechanism, leading to excessive calorie intake and weight gain. In addition, a theory on energy homeostasis has been proposed as another possible mechanism for speculated weight gain from low-calorie sweeteners. Energy compensation, which is the adjustment of energy intake on subsequent meals based on amount of energy consumed on the prior meal, may be disrupted when caloric sweeteners is replaced with low-calorie sweeteners (283). Because low-calorie sweeteners provide only negligible amounts of energy, an upward compensation by increasing intake on subsequent meals is expected. More importantly, some worry that use of low-calorie sweeteners could lead people to believe that they could consume more of other foods to compensate for lower energy from low-calorie sweetened foods and beverages (280). Overall, there is no current evidence on humans to validate any of these theories. However it is clear that inappropriate use of low calorie nutritive or non-nutritive sweeteners that leads those attempting to control their weights to throw all caution in consumption to the winds will nullify any potentially positive effects that they might have.

Low calorie sweeteners have no or modest effects on weight loss and weight maintenance unless accompanied by a hypocaloric diet

A number of studies have been conducted or are now in progress to determine the effects of low-calorie sweetened beverages on
weight changes and metabolic health effects in adults and children in adults, epidemiological studies of children and adolescents are mixed, some showing associations of low calorie sweeteners are associated with increased body weight (319). Two studies (the cross-sectional National Health and Nutrition Examination Survey (NHANES) and the semi-longitudinal San Antonio Heart Study), found positive associations between the use of non-nutritive sweeteners and an increase in body mass index (BMI) (268). However, the design was such that it is not clear if obese people used more low-calorie sweeteners (more likely) or whether low-calorie sweeteners caused them to be obese (less likely). Another longitudinal cohort study found a positive correlation between increased consumption of diet soda and BMI z-scores after two years in 164 elementary school aged children (284). However, the study design again was not such that it could demonstrate a causal relationship between the two, possibly the effects were again due to reverse confounding (e.g. the obese who are most likely to gain weight were those who also used more diet products). Attributes of causal relationships such as strength of association, temporal relationship, consistency of findings, biological plausibility, and strong dose-response relationship, need to be considered when epidemiologic studies of low-calorie sweetener consumption and weight gain are examined. In the few small randomized trials of overweight and obese children, low calorie sweeteners do not or only minimally decrease weight and body mass index (320, 321, 322). Therefore, although there is no strong evidence that use of low-calorie non-nutritive sweeteners (especially in beverages) increases weight, there is also little evidence that it decreases it substantially. However, adding several interventions, such as was done in the 6 month America on the Move study, in which 192 families with at least 1 overweight child were randomized to either an intervention involving increasing physical activity by 2000 steps a day and decreasing sugar by 100 Calories per day with a low calorie sweetener or to a control group which involved only self-monitoring, the two interventions together did show small but greater changes in body mass index for age among the children at month 6 (323).
Another theory to explain the purported association between low-calories sweeteners and weight gain is that lower carbohydrate intake may result in higher fat intake. A reduction in carbohydrate intake by replacing sucrose with low-calorie sweetener is claimed to lead to a higher proportion of energy coming from fat, which in turn may lead to weight gain. However, accumulating evidence does not support the claim that macronutrient shifts occur from the replacement of low-calorie sweeteners and an increased fat intake in the diet (280).
Another potential, but unproven theory based on a study in rats linking low-calorie sweeteners and weight gain is that the gut microflora are altered when they are exposed to low-calories sweeteners. Changes in gut microflora might then trigger inflammatory pathways; promoting insulin resistance, fat accumulation, and weight gain in the individual (286). There is no evidence in humans to support this claim, and the evidence in experimental animals is mixed at best..

Development of a “sweet tooth” due to low calorie sweeteners?

Artificially sweetened and calorically sweetened foods and beverages are claimed to increase the preference for a sweet taste, with an increase in consumption of artificial sweetened beverages may change taste preferences toward sweet foods, especially in children (268). This preference is feared by some to bring about the replacement of healthful foods with sweets, and thus may be associated with lower diet quality in children. However, at present little evidence exists that this is so. In addition, repeated exposure to sweetness could lead to an increased acceptance of sweet sensation as a result of learned behavior and the exposure to sweetness in low-calorie sweetener might establish preference to sweetness in the same manner as other sugars (280).

Low-calorie non-nutritive sweeteners have been shown to activate sweet-taste receptors in the gut in the same manner as sugar does in animal studies. T1R2 and T1R3 are transmembrane sweet-taste receptor proteins found in the gut of both rodents and humans (286, 287). Compounds with a sweet-taste (e.g. sugar and low-calorie sweeteners) bind to these receptors, stimulating signaling pathway to the brain in experimental animals. This leads to changes in electrophysiological patterns in the brain, resulting in preference of sweetness. At present, the evidence that low-calorie sweeteners encourage or exacerbate sweet tooth remains scattered. More research is needed.

What can be said with certainty is that infants and young children do not require low-calorie sweeteners although they are judged as safe by the FDA. The American Dietetic Association (ADA) suggests that parents of children less than 2 years of age discuss use of those products with their pediatricians to ensure that childrens’ needs are met (244). Concerns about ensuring that children and adolescents learn good food habits have led some individuals and expert groups to recommend that schools restrict the use of calorically sweetened beverages and high-calorie, low nutrient density sweet foods that are also high in added sugars (288). However, some of those reports have also recommended against the sale of low-calorie sweetened beverages in school, not for safety reasons, but to foster nutrition education. The rationale for doing so is not entirely clear, but nonetheless, many elementary and secondary schools now do not permit non-nutritive sweetened or calorically sweetened beverages in schools. Research is needed to determine if such a prohibition has intended effects on nutrition education, development of a “sweet tooth”, dental caries, weight gain or maintenance, overall intakes of sugar, added sugar, or food energy, or other objective standpoints. Also, unintended effects, such as more extensive overall use of the products as gestures of defiance must be assessed.

Metabolic Syndrome

Some large prospective cohort studies found associations between intakes of low-calorie sweeteners and incidence of metabolic syndrome, but the designs were such that cause and effect could not be demonstrated (289). Proponents that such a link is causal suggest that the association is due to deregulation of glucose homeostasis caused by the use of artificial sweeteners. Another, simpler, explanation is a reverse causation. One study on consumption of diet soda in young healthy volunteers before an oral glucose challenge showed an increase in secretion of GLP-1, the anti-hyperglycemic hormone released by intestinal cells, which can alter gastric emptying and secretion of insulin. Whether the low-calorie sweeteners in fact had these effects is not yet clear; the study has not been replicated. The concern raised is that consumption of artificial sweeteners together with foods and drinks containing sugar may lead to increased GLP-1 and insulin secretion, which may result in more rapid sugar absorption and perhaps in turn, could influence blood sugar levels. However, at present, evidence is lacking that this is the case.

Evidence from in vitro and rat studies suggests that low-calorie sweeteners stimulate the sweet-taste receptors in the gut, causing the up-regulation of transporters, sodium-dependent glucose transporter (SGLT 1) and glucose transporter 2 (GLUT 2), on the apical membrane of the small intestine to increase glucose uptake. In addition, low-calorie sweeteners (especially sucralose and saccharin) may also stimulate release of incretin (GLP-1), a gut hormone that stimulates insulin release after ingestion of glucose. This cascade of intestinal uptake transporters, incretin, and insulin release may disrupt glucose homeostasis (286, 287). However, extensive number of studies conducted in humans showed no such effect. Overall, there is no consistent evidence in humans that low-calorie non-nutritive or nutritive sweeteners have adverse effects on insulin release and blood glucose homeostasis.

9.9.3.7 SUMMARY AND RECOMMENDATIONS

None of the low-calorie sweeteners permitted by FDA on the US market are necessary in human diets or for human health. However, some consumers enjoy the sweet taste of nutritive or non-nutritive low calorie sweeteners and there is no reason to discourage their use on safety or health grounds.
All FDA-approved sweeteners are safe for use by the general population, pregnant women (except phenylketonurics), and children (except phenylketonurics) as doses below the Acceptable Daily Intake (ADI). For pregnant women (other than those with PKU) the American Academy of Pediatrics has stated that aspartame is safe for pregnant women and their developing infants. There is also evidence that acesulfame K, sucralose, and the sugar alcohols are safe for pregnant women in small amounts. Little research has been conducted on the safety of saccharin and stevia in pregnant women.
None of the low-calorie nutritive or non-nutritive sweeteners are perfect substitutes for sugar from the standpoint of taste, mouth feel, and functionality in food processing and preparation, but they are acceptable to many people.
Most of the nutritive and non-nutritive low-calorie sweeteners are non-cariogenic. Xylitol also appears to have an anti-cariogenic property as well, enhancing remineralization of tooth enamel.
The best-documented adverse effects with low-calorie sweeteners are from poorly absorbed nutritive low-calorie sweeteners (sugar alcohols) that have laxative effects when consumed in large amounts. A theoretical hazard exists for aspartame with very heavy use of individuals with phenylketonuria (PKU). Other health concerns, such as the fostering of a “sweet tooth” that leads to excessive energy intakes are poorly documented.
Low calorie sweeteners may be helpful in weight control if they are substituted for some of the caloric sweeteners in the diet and calorie intakes are also reduced. Otherwise, they appear to have little effect. Their use alone without healthful eating, physical activity, and behavior change is unlikely to be helpful in weight management.
Low-calorie sweeteners contribute little or nothing to the glycemic response and as such may have advantages over caloric sweeteners for individuals who need to control their blood sugar levels.
There is no current scientific reason to recommend against the use of low calorie sweeteners for those who are trying to lose weight and wish to use them.
For patients or other users who are concerned about excessive intakes of nutritive or non-nutritive low calorie sweeteners, the best advice is to use several different types of low-calorie sweeteners so that doses of any one compound are low and to always stay below the ADI, or to not use them.

Table 20 is a description of nutritive and nonnutritive low-calorie sweeteners approved by the FDA or recognized as generally recognized as Safe (GRAS). The table describes the following for each non-nutritive and nutritive sweetener; brand name, definition, characteristics, metabolism and excretion, chemical structures, sweetness, calories, ADI, year approved, uses, health benefits, health concerns and additional comments.

Table 20. Nutritive and Non-nutritive low-calorie sweeteners approved by FDA or recognized as Generally Recognized as Safe (GRAS) (242-291).
Non-nutritive sweeteners								Nutritive Sweeteners
Names	Aspartame	Acesulfame-K	Saccharin	Sucralose	Neotame	Advantame	Steviosides	Mannitol	Xylitol	Sorbitol	Erythritol
Brand names	NutraSweet®, Equal®, others	Sunett®, Sweet One®	Sweet’N Low®, Sweet Twin, Sugar Twin®, Necta Sweet®	Splenda®	Used as ingredient in food products.	Used as an ingredient in food and beverage products	Stevia®, Truvia™, Sun Crystals®, PureVia™, Sweetleaf Sweetener™	Used as ingredient in food products.	XyloSweet	Used as ingredient in food products.	Zerose
Definition	Synthetic sweetener composed of aspartic acid and phenylalanine.	A combination of an organic acid and potassium.	Synthetic sweetener in forms of sodium or calcium saccharin.	A sugar derivative by replacing 3 hydroxyl groups with 3 chlorine atoms on the sugar molecule.	Dipeptide methyl ester derived from aspartic acids and phenylalanine.	Synthetic sweetener produced in a 3-step process that ultimately combines aspartame and HMPA	Derived from the leaves of Stevia rebaudiana plant in South America. Known as “sweet leaf.”	A hexose alcohol extracted from seaweed.	An intermediate product of carbohydrate metabolism from xylan-containing plants.	A hexose alcohol from hydrogenation of glucose and fructose with nickel catalyst.	A tetrose alcohol derived from the cultivation of yeast-like fungi on glucose.
Characteristics	Loses sweetness with high heat.	Highly heat stable for cooking and baking. Metallic aftertaste.	Highly heat stable for cooking and baking. Bitter metallic aftertaste.	Highly heat stable for cooking and baking.	Highly heat stable for cooking and baking. Clean sweet sucrose-like taste.	Heat Stable for cooking and baking. Clean sweet sucrose like taste. Ultra high potency.	Heat stable. Licorice aftertaste. Enhances sweet and savory flavors. Lacks bulking property.	Heat stable. High melting point. Non-hygroscopic(does not pick up moisture).	Sweetest of sugar alcohols. Quickly dissolves. Produces cooling effect in the mouth.	Heat stable and highly soluble. Does not cause browning. Humectant (retain moisture).	Very water-soluble. Non-hygroscopic.
Non-nutritive sweeteners								Nutritive Sweeteners
Metabolism and Excretion	Broken down into aspartic acid, phenylalanine, and methanol upon digestion. All compounds are metabolized normally, except in individuals with PKU.	Not metabolized and excreted unchanged by kidneys.	Not metabolized and excreted unchanged by kidneys.	Not randomized and excreted by the kidneys and in feces.	Partially absorbed and excreted in feces and urine.		Not absorbed in small intestine. Degraded into steviol by bacteria in the colon, where it is absorbed. Excreted in the feces and urine.	25% is absorbed and excreted in the urine. Unabsorbed portion is fermented by colonic bacteria.	50% absorbed and excreted. Unabsorbed portion is fermented by colonic bacteria.	25% is absorbed and excreted in the urine. Unabsorbed portion is fermented by colonic bacteria.	90% is absorbed. Rapidly excreted in the urine and feces within 24 hours.
Relative sweetness compared to sucrose*	180	200	300	600	7000 - 13000	20000	200 - 300	0.5 - 0.7	1	0.5 - 0.7	0.6 - 0.8
Kcal/g	4	0	0	0	0	0	0	1.6	2,4	2,6	0.2
ADI (mg/kg/d) **	50	15	5	5	18mg / NA	1970 mg/day	0-4 (as steviol)	Not specified.	Not specified.	Not specified.	Not specified.
ADI for 70kg person / Cans of soda equivalent	3500mg / 28	1050mg / 21	350mg / 4	350mg / 6	18mg / NA	1970 mg/ NA	0 – 280mg / 5	NA / NA	NA / NA	NA / NA	NA / NA
Year of approval by FDA and as GRAS.	1981	1988	Prior to 1958. Reapproved again in 2000.	1998	2002	2014	GRAS in 2008	1986	1983	GRAS 1982	GRAS in 2001
Chemical Structures	Aspartame	Acesulfa me-K	Saccharin	Sucralose	Neotame	Advantame	Steviosides	Mannitol	Xylitol	Sorbitol	Erythrit
Non-nutritive sweeteners								Nutritive Sweeteners
Uses	Tabletop sweetener, ingredients in foods and diet soft drinks. Limited use in bakery products.	Tabletop sweeteners, baked goods, frozen desserts, candies, beverages, cough drops, and breath mints.	Tabletop sweetener, soft drinks, baked goods, jams, chewing gum, canned fruit, candy, dessert toppings, salad dressings.	Tabletop sweetener, beverages, chewing gum, frozen desserts, fruit juices, gelatins.	Flavor enhancer, baked goods, soft drinks, chewing gum, frozen desserts, jams, puddings, gelatins, processed fruits.	Flavor enhancer, baked goods, soft drinks, chewing gum, frozen desserts, jams, puddings, gelatins, processed fruits.	Tabletop sweetener, juices, tea beverages. (Used extensively in Japan for pickles, dried seafoods, and confections).	Dusting powder for chewing gum, ingredient in chocolate-flavored coating agents for ice cream and confections.	Chewing gum, hard candy, oral health products, cough syrups and cough drops, children’s chewable multivitamins, foods for special dietary needs.	Sugar-free candies, chewing gums, frozen desserts, pastries	Bulk sweetener in diet food products, candies, beverages, fat-based creams, chewing gums, confection, yogurt.
Health benefits	Virtually calorie free.	Calorie free.	Calorie free.	Calorie free.	Calorie free.	Calorie free.	Calorie free. Claimed to have a hypoglycemic effect.	Low calorie content. Non-cariogenic. Low glycemic response.	Low calorie content. Reduces dental plaque and caries and may promote tooth remineralization. Low glycemic response.	Low calorie content. Slow absorption and metabolism independently of insulin might benefit for diabetics.	Calorie free. Unlikely to have a laxative effect. Non-cariogenic.. Low glycemic response.
Non-nutritive sweeteners								Nutritive Sweeteners
Health concerns		All should be used at levels below the ADI.						Strong laxative effect at >20 mg/day.	Strong laxative (> 50 mg/day) and also diuretic effects. Flatulence and diarrhea.
Comment	Requires a label that product contains phenylalanine.				Does not require a label for phenylalanine content due to negligible amount used and low availability of phenylalanine from the neotame.			Requires a warning label for a possible laxative effect.		Requires a warning label for a possible laxative effect.
Relative sweetness as compared to sucrose (table sugar). 1= reference value which is the sweetness of sucrose. * ADI = Acceptable Daily Intake *** Other non-nutritive low-calorie sweeteners (Alitame, Thaumatin, Neohesteridine, and Glycyrrhizin) are not yet approved as both sweeteners and as GRAS in the US. See text for details.http://beverageinstitute.org/

9.10 Energy Density

Energy density, or caloric density, is defined as the calories provided per unit weight of food eaten (such as calories/gram). When the composition of a diet of usual foods is decreased in fat, the energy density of the diet tends to fall since the total weight of food consumed remains constant, or may increase (196-198) . Some of the beneficial effects of low fat diets in weight loss and maintenance may be due, at least in part, to low energy-density, which may promote satiety. The premise is that if foods bring more weight than calories into the body, gastric distention and intestinal bulking may promote fullness. For example, foods high in water and/or fiber tend to have low energy density, with some that are very high in volume, such as unbuttered popcorn. Their inclusion in a weight reduction diet is advocated by some experts for this reason (10), and because they might increase overall dietary quality (199). As will be discussed later, some evidence suggests that low-energy density, high-volume diets may help people ingest fewer calories and thus may assist with weight loss, although longer-term research is needed (200; 201;199).

9.11 Volume of Food

Low energy density is the basis of the “Volumetrics” diet put forth by Dr. Barbara Rolls. This diet is high in fruits, vegetables, cooked whole grains and fibrous foods, which are high in water and bulk, to add volume for satiety without extra calories (285). In a one year study completed by 71 obese women, the low energy density diet group showed slightly better weight loss than a low fat diet group, and reported lower hunger (324). Cross-sectional work has shown that people who maintain large
amounts of weight loss over years tend to eat a lower energy density diet than normal weight or obese subjects (201). Some other diets tend to be low in energy density without specifically targeting energy density, through their inclusion of fruits, vegetables, and fibrous foods (199).

10 Available Programs

According to the U.S. Food and Drug Administration (FDA), Americans spent an estimated $30 billion in 1992 on diet and weight loss programs. Market data, an independent market research and consulting firm, has estimated annual spending on diet and weight loss programs to have reached $61 billion by 2013 (203). The number of products and programs available to consumers is essentially endless, and the quality of what is available can be questionable. Therefore, patients should be advised to research programs or products they are interested in, and consult with a physician before utilizing any commercial weight loss option.

10.1 Registered Dietitians: Dietetic Advice and Individualized Eating Plans

A registered dietitian is a food and nutrition expert who has met academic and professional requirements including:

A bachelor’s degree at an accredited university, approved by the Commission on Accreditation for Dietetics Education (CADE) of the American Dietetic Association (ADA).
Completed an accredited, supervised, experiential practice program (dietetic internship) at a health-care facility, community agency, and/or foodservice corporation, including at least 900 hours of hands-on experience.
Pass a national examination administered by the Commission on Dietetic Registration (CDR).
Complete continuing professional education requirements to maintain registration.

Many physicians lack the time that obese patients require for successful weight control therapy. Referral for dietary counseling to a registered dietitian (RD) is useful for many patients, particularly those who have comorbidities that also require medical nutrition therapy. Many registered dietitians also hold certifications in specialized areas of practice, including weight management, providing additional expertise in the management and treatment of obesity (203). They are able to understand more complicated therapeutic dietary recommendations that many patients, especially those with comorbidities, require. Practicing registered dietitians in your area can be located using the website for the Academy of Nutrition and Dietetics at http://www.eatright.org/.

10.1.1 Available Programs

There are over 67,000 registered dietitians in the United States, practicing in hospitals, outpatient centers, health centers, the community, and in private practices, among many other areas of expertise (204). Patients can see a registered dietitian via patient referrals from physicians for a variety of health problems that require dietary modification, or by self-made appointments. Depending on the area of dietary modification/treatment, health insurance may, or may not, cover the services provided, so patients need to check with their insurance providers before scheduling an appointment. Some formal weight control programs staffed by dietitians are available in hospitals and health centers, where individual counseling is also available.

10.1.2 Candidates for Care

Dietary advice of a general nature is not enough for patients who have multiple comorbidities requiring medical nutrition therapy (e.g., diabetes, hypertension, coronary artery disease, gastrointestinal disorders, etc.), those on multiple medications, and those with complex and involved health problems that have dietary implications. These patients are prime candidates for dietetic therapy with a dietitian. Patients who have had poor outcomes in weight control efforts on their own, who have special dietary needs or preferences, and who need extensive education and assistance are also particularly likely to benefit.

10.1.3 Advantages

Registered dietitians are able to read and interpret medical records and are equipped to adopt weight loss prescriptions to the particular needs of patients. Their knowledge of food habits, food preparation, and food products on the market makes them an excellent resource for helping patients to adopt the general weight control prescription to ta patient’s particular circumstances. A particular advantage of dietetic involvement in patient care is that dietitians often work in medical settings and have access to patient charts, as well as the ability to consult with other health professionals. Registered dietitians are helpful in treating patients on multiple medications, on very-low-calorie diets, and post-gastric bypass counseling. Some dietitians have advanced certification in weight management, and are especially well equipped to counsel patients with complex and involved medical problems. Some insurance companies and health maintenance organizations may pay for obesity treatment when it is part of a larger therapeutic program for conditions such as diabetes if it involves a dietitian who is a certified Medicare provider. Patients should check with their insurance providers about reimbursement.

10.1.4 Disadvantages

The patient’s out-of-pocket costs for dietary counseling from a registered dietitian vary, depending on insurance coverage and the comorbidities that need to be treated/addressed. It is important to note that personal trainers and/or nutritionists do not necessarily have the credentials to be counseling patients on nutrition and dieting. This is particularly true of patients who have comorbidities. . Providers should encourage patients seeking counseling on their own to look for the “registered dietitian” credential.

10.1.5 Safety and Effectiveness of Therapy

Registered dietitians are health professionals who go through extensive schooling and training and are registered in a national registry with specific standards. Licensure is also required in 31 states. Registered dietitians, therefore, have medical, legal, and ethical obligations to their patients. Their own education includes formal educational requirements of at least a baccalaureate degree, a dietetic internship, supervised clinical training, a registration exam, and mandatory continuing education. Dietitians are trained to read medical charts, to work with physicians and other allied health professionals, and to alert physicians when untoward events arise. Thus, their recommendations regarding weight loss are likely to be safe and evidenced-based. The effectiveness of dietetic counseling, like that of physician counseling for weight control, has seldom been evaluated.

10.2 Commercial Weight Loss Programs

Commercial non-medical weight control programs are popular and widely available in the United States and Canada. They will be discussed in detail in the following section.

10.2.1 Available Programs

Commercial programs include large chains such as Weight Watchers®, Jenny Craig®, LA Weight Loss Centers®, Nutrisystem®, and many regional ventures. These programs vary, but generally include advice on a structured low calorie diet, exercise, lifestyle modification coupled with group support and/or individual counseling. Oftentimes, there are options for delivery of pre-portioned reduced-calorie meals. Usually the program is administered by a layperson trained by the program who is often a successful program graduate. However, laypersons trained by the company and degree-trained professionals (such as dietitians) may also be on their staff. It should be noted that these programs do not provide physician supervision although they usually require physician sign-off before involvement (105). All of these programs are for-profit entities and charge fees (196). With the growth of the Internet, many programs now offer online support as an adjunct or replacement to more traditional in-person individual or group counseling. A sample of some popular commercial programs is outlined below in Table 21.

Table 21. Popular Commercial Programs
Product	Comments
Jenny Craig® (Nestle Nutrition®) www.jennycraig.com	A commercial program where dieters are paired with trained consultants, often program graduates, who help set goals, plan weekly meal plans, and provide support through in person and phone meetings. The program also includes home-delivered pre-portioned food that provided 3 meals and a dessert or snack each day. The meals are designed to promote 1-2 lbs of weight loss each week using portion control. The plan starts at $19 per month, plus the cost of food, which ranges from $16-$23 per day. A full line of products is also available to be purchased outside the official program in retail outlets. Options in this line range from Complete Meals to Café Steamers. Meals are between 180-410 calories and are available at most local grocers. There are also desert options. These products fulfill American Heart Association heart checklist program criteria, and are lower in sodium than some other brands
Nutrisystem® (Nutrisystem Inc. ®) www.nutrisytem.com	Participants in the program receive all meals, snacks, and desserts via home delivery. Foods may be chosen a la carte or according to a meal plan with predetermined food choices. Various plans are offered for men and women in different categories: basic for the budget conscious, core for added convenience and variety, select for the most variety, diabetic for diabetics, and vegetarian for vegetarians. Prices vary depending on the plan, but are expensive. One month on the program can cost between $300 and $500, and may be more expensive. The program also includes access to an online community and phone counseling. 5-day weight loss kits are also available in retailers such as Wal-Mart® and Costco® for around $50.00.
Weight Watchers® www.weightwatchers.com	Weight Watchers uses a group support focus to promote weight loss through attendance at weekly meetings, where members support each other, discuss challenges and successes, and weigh-in. Weight loss is achieved by teaching dieters how to subscribe points to a variety of different foods and eat within a certain point budget each day. For around $10 per week, dieters can attend meetings and have access to online tools and a mobile app. There is also an online-only self-help version of the Weight Watchers® program which provides a diet plan and fitness information with exercises. Cost is currently around $20 per month, with an approximately $30 sign-up fee. However, the often run cost-saving promotions for those willing to sign-up for at least 3 months.
LA Weight Loss Centers® www.laweightloss.com	A 3-part plan that provides meal plans with recipes, snack bars, and dietary supplements. Other tools available include educational material, food diaries, and specialized plates and containers to aid in portion control. The silver plan cost between $150-$200 per month, and does not include supplements, while the gold plan costs between $200-$250 per month and offers supplements designed by the company called “nutritionals”. Support from counselors is available in-center and online.
Ediets.com www.ediets.com	Provides a personalized reducing diet and food list, fitness information, healthy recipes, social networking community, and charts and dieting tools. There are 3 categories of diet plan currently available: the “Vitabot plan”, the “Holly Madison Diet,” and the “Nutrihand plan.” Cost is currently $9.95 per month. Meal delivery options are also available for $30 - $40 per day through “The Chef’s Diet,” which uses a 40-30-30 ratio of carbohydrate, protein, and fat, respectively, and between 1300-1500 calories per day. This option includes 3 meals per day and 2 snacks
Diets.comwww.diets.com	A nutrition and health website with tools to aid in weight loss, healthy living, and wellness. Basic membership is free and includes access to online support groups, articles, and useful tracking tools. However, premium membership, including all of the above in conjunction with a customized diet plan, personalized exercise plan and coping plan, personalized “expert” advice, and individualized weekly self-checklists to help keep dieters on track, is available for around $40 per month, with those willing to sign-up for 6 months getting the largest discount at just over $14 per month.

10.2.2 Candidates

Overweight and moderately obese persons with few risk factors and few comorbidities are good candidates for these programs. Those who find that they need continued motivation, monitoring, and social support with a structured regimen may particularly benefit from one of these programs.

10.2.3 Appropriate Use

These programs are not substitutes for physician concern for or medical monitoring of his or her patients’ weights. They are most successful when the patient’s personal physician continues to provide encouragement and supervision because most commercial weight loss programs provide no or very little physician supervision. The commercial programs are not equipped to deal with patients with multiple involved comorbidities of either a medical or psychological nature. Patients with complex medical issues are better treated by a program and therapists who are more closely connected to the health care system where medical charts and other patient-specific information is available. Registered dietitians and specialized weight control programs operated by medical facilities are more appropriate options in this population.

10.2.4 Advantages

Most major established commercial chains offer well-crafted, nutritionally adequate, and behaviorally sound programs that, overall, are reasonable therapies. Classes are often held in places of employment or neighborhood centers that are conveniently located. Weight Watchers® offers frozen entrées and other weight control products that are integrated into the program and available in supermarkets, making adherence easier. Jenny Craig® and LA Weight Loss Centers® also offer frozen entrees and various weight control products; however, these are only available through their stores. On the Nutrisystem® program, the customer must eat only Nutrisystem® food for a defined period based on individual needs. Nutrisystem® sends all food items including snacks to the customer via mail.
In a multicenter, randomized, two-year study of 423 subjects with a BMI of 27 to 40 kilogram/m2 it was shown that a structured commercial weight-loss program was more likely to be effective for managing moderately overweight patients than brief counseling and self-help (205). Individuals were randomly assigned to either a self-help program, consisting of two 20-minute sessions with a nutritionist and provision of printed materials and other self-help resources, or to attendance at meetings of a commercial program (Weight Watchers®). After 26 weeks subjects in the commercial weight-loss program had greater decreases in body weight, BMI, mean waist circumference, and fat mass (205). It is important to discuss commercial program options with individuals so they know their options, but only with individuals who are plausible candidates.
Since it was founded in 1997, most of the large commercial programs have joined the Partnership for Healthy Weight Management, a voluntary association. Members provide, on a voluntary basis, publicly available information to help potential participants meet their needs. Criteria for membership require that programs disclose staff qualifications, essential components of the program, the risks associated with overweight and obesity, other details about the provider’s program or product, and program costs.

10.2.5 Disadvantages

Because they are profit-driven business, the main objective of a commercial weight loss programs may not be driven by patient care. Although most programs require physician approval before participants can enroll, there is no guarantee of the quality of the health assessment that has been carried out prior to enrollment. For some individuals, especially those at very high risk, more intensive medical supervision may be required. The cost of the programs is another obstacle. Many of the poor who are obese do not have the resources to purchase these services and products, even though they might benefit from them. Discounts or waivers of fees for those in financial hardship are rarely available.
Statistics are rarely kept on success rates or long-term adherence. Another problem is maintenance of lost weight and preventing relapse. The companies have become more active in developing programs catering to those who have lost weight to help them maintain their losses in recent years, but incentives to patients for staying in maintenance programs may still not be sufficient. Additionally, very few high-quality studies have assessed the efficacy of commercial weight loss programs and the ones that do provide the best-case scenario for results—as they do not account for participants who have dropped out of the program (206). The only program that has published high-quality studies to date is Weight Watchers®. The best study on Weight Watchers® determined that participants lost 5% of their initial body weight (about 10 pounds) in 6 months and kept off 3% (about five pounds) at two years (206).

10.2.6 Effectiveness and Safety

The major firms provide programs and products that are safe for patients without major comorbidities when directions are followed. However, in spite of the fact that millions of Americans have purchased these services, their effectiveness in bringing about weight loss or sustaining lower weights has rarely been studied with scientific rigor (105).

10.3 Formulas and Meal Replacements

In addition to commercial weight loss programs, many meal replacement and formula products for weight control are now available. Patients can purchase these products on their own in supermarkets, drug stores, and online. Unlike very low-calorie diet formulas, which are medical foods that are usually provided as part of a medically supervised treatment program (see Table 13), these products can be purchased by anyone.

10.3.1 Available Products

Meal replacements now include not only powders like Slim-Fast® that are mixed with milk or other liquids, but drinks, bars, and frozen entrees. Formulations and nutrient content vary. Most liquid meal replacement products provide about 220 calories per serving and are relatively high in protein, vitamins and minerals, but low in fat (see Table 22 for examples of over-the-counter, ready-to-drink, liquid meal replacements). The health bars and frozen entrees vary in their caloric content, but are generally between 200 and 400 calories, and have more complete profile of nutrients. The entrees include offerings such as Lean Cuisine®, Healthy Choice®, and Smart Ones®, among others (see Table 23 for product listing). All of these pre-packaged entrees share characteristics such as discrete portion sizes that are relatively low in calories (usually 300 calories or less). Generally, all frozen entrees are high in sodium, with at least 500 mg of sodium per serving. Smart Ones® is manufactured by HJ Heinz, and is closely allied with the Weight Watchers® commercial diet program. Its packages are prepared to fit into the food plans for the Weight Watchers® program. All of the meal replacement products are designed to be eaten with additions of conventional foods that supply dietary fiber, other nutrients, additional calories and fluids.

Table 22. Examples of Over the Counter, Ready to Drink, Liquid Meal Replacements for Weight Loss
	Total calories	Size	% Carbohydrate	%Protein	%Fat
GNC® Total LeanTM Lean Shake TM (GNC®)	170	14 fl oz	6	25	6
EAS® Myoplex® Lite (Abbott®)	170	11 fl oz	20	20	2
Slim-Fast® Protein Meal Shakes (Unilever®)	180	10 fl oz	4	20	9
Atkins™ Advantage® Shakes (Atkins Nutritionals®)	160	11 fl oz	6	15	9
Glucerna® Hunger SmartTM Shake (Abbott®)	180	11.5 fl oz	16	15	8
Carnation® Breakfast Essentials™ No Sugar Added Complete Nutritional Drink (Ready-to-Drink) (Nestle Nutrition®)	250	11 fl oz	16	13	5
Note – Glucerna is formulated for individuals with diabetes or prediabetes

Table 23. Popular Frozen Entrees
Product	Comments
Healthy Choice®(ConAgra Foods®)	A full line of products from Complete Meals to Café Steamers. Meals are between 180-410 calories and are available at most local grocers. There are also desert options. These products fulfill American Heart Association heart checklist program criteria and are lower in sodium than some other brands
Kashi® Frozen Entrées(Kashi®)	A variety of different entrée options are available, many of which are vegetarian. Typically, these entrees range from 250 to 400 calories. They are available in local grocery stores.
Lean Cuisine®(Nestle Nutrition ®)	A wide range of entrees from Panini sandwiches to lasagna. Entrées, including breakfast options, are between 140-400 calories, and are available at many grocery stores.
Smart Ones®(Heinz®)	Smart Ones® products are associated with the Weight Watchers® program, and available at many grocery stores. Entrees range from 180 to 310 calories, and packaging also includes the Weight Watchers® points associated with each item. A small tossed salad and/or fruit may be added to make the meal more complete. There are also snack, desert, and breakfast options available.

10.3.2 Candidates

Individuals who are healthy but moderately overweight (BMI 25-30) and who wish to lose less than 5% of their body weight or who wish to use these products for one meal a day to assist in their weight maintenance efforts may find these products helpful. The products provide an easily prepared, generally nutritious, and relatively modest caloric load that can satisfy hunger. For those who are susceptible to environmental triggers (such as being involved in meal preparation or eating in cafeterias or fast food restaurants) and respond by overeating, these products offer a safe and palatable option that lessens temptation.

10.3.3 Appropriate Use

Portion controlled liquid meal replacements such as Slim-Fast® or Shakeology (and many other products) are recommended for two meals and a snack with a small meal of conventional foods and low or no calorie beverages. They should not be used as the sole source of nourishment on a diet. The entrée choices are suitable for meals, but their use for multiple meals a day should be cautioned in patients for whom sodium consumption is a concern.

10.3.4 Advantages

The main advantages of meal replacements are built-in portion and calorie control, widespread availability, convenience composition that is fairly micronutrient dense while remaining low in calories, ease of preparation, and for some of the dry or canned products, portability. Costs of the meal replacements are reasonable, and can simplify food choice decisions. They are lower in calories than many snack or restaurant foods that people who are eating away from home might otherwise consume. They are also convenient, rapidly and easily prepared, and can be eaten anywhere, allowing eaters to avoid “high risk” eating environments.

10.3.5 Disadvantages

The major disadvantages of these products are their cost, monotony, and limited variety. From a nutritional standpoint, the products vary, but are often quite high in sodium (600 plus milligrams per serving). Only Healthy Choice® is low in calories, saturated fat, and also in sodium. As with most strategies, they are ineffective unless they are used as part of an overall low calorie eating plan. If they are used as sole sources of food they would be nutritionally inadequate not only in energy, but several other nutrients and water. Additionally, they might not provide a patient trying to lose weight with practice in planning and preparing their own healthy low-calorie meals for a lifetime of healthy weight management. However, there is little evidence on this part.

10.3.6 Effectiveness and Safety

These products may be nutritionally inadequate when they are used as the sole sources of food and fluids for many weeks. When the products are used according to directions on the label or in package inserts, they are safe (71) . When used as part of a weight loss program these single meal replacements are effective during the weight loss phase (198). They are also valuable additions in the weight maintenance phase, often because the meal replacements provide a low set number of calories in an easy-to-fix-entrée (5), with control over portion size (207).

10.4 Weight Loss Books and Manuals

In addition to weight loss products, Table 24 provides some examples of popular diet books. Books are difficult to use on one’s own because there is little reinforcement. The quality of self-help books on weight control ranges from the sublime to the ridiculous. Among the better, older books currently on the market are the LEARN® Program for Weight Management, which is a sound 15 week course that is usually administered within a treatment program (208). The book is effective when it is part of the treatment program. However, the charges for such a program are considerable, the program is not available in all parts of the country, the effectiveness of self-directed efforts using the book by itself has not been evaluated, and the dietary advice is often vague. Another good book is Volumetrics by Barbara Rolls PhD (209), which encourages a diet based on foods that have a low energy density, meaning that they contain few calories per gram of weight. Dr. Rolls’ research has shown that foods with large volume but few calories can provide satiety while helping individuals avoid over-consumption of energy. Such foods are usually high in water and fiber, while low in fat. Although the long-term efficacy of this specific diet has yet to be affirmed, the diet is rich in fruits, vegetables, and other healthful foods (209). The bottom line on diet books is that with few exceptions, the dieter is sure to lose his or her money , but whether weight is lost or not is less certain. Moreover, in spite of the hype there is little evidence that, aside from the few books mentioned above, that the diets “work” and that the authors have discovered a unique new scientific principle that causes weight loss.

Table 21. Popular Diet Programs and Books (46)
Diet	Brief description	Average Calories Per Day	Composition % of Calories			Type of Diet
			%CHO	%Protein	%Fat
5-Factor DietHarley PasternakBallantine Books, 2009	5 week plan, 5 meals per day, 5 minute preparation time per meal, recipes with only 5 ingredients, 5 cheat days in 5 weeks, and 25 minute workouts 5 days a week for 5 weeks	1300	58	32	10	Weight Loss
The Abs DietDavid Zinczenko, Editor-in-Chief of Men’s HealthRodale Books, 2005	This diet is based on foundation foods that conform to the acronym Abs Diet Power : A lmonds and other nuts B eans and legumes S pinach and green vegetables D airy (fat free or low fat) I nstant Oatmeal E ggs T urkey and lean meats P eanut butter (natural and sugar free) O live oil W hole-grain breads and cereals E xtra protein (whey powder) R aspberries and other berries	1700	45	25	30	Weight Maintenance
Atkins™ for LifeDr. Robert C. Atkins™, MDSt. Martin's Griffin, 2004	A low carbohydrate plan for those who have lost weight with the original Atkins™ Diet. Dieters are advised to cut back on carbohydrates if weight loss stops.	Phase 1: 1540	24	21	55	Weight Maintenance
		Phase 2: 1970	22	22	56	Weight Maintenance
		Phase 3: 2310Pre-Maintenance	29	19	52	Weight Maintenance
		Phase 4: 2050Lifetime Maintenance	35	20	44	Weight Maintenance
Eat Right 4 Your Type (The Blood Type Diet)Dr. Peter J. D’AdamoPutnam Adult, 1996	Based on the idea that tailoring one’s diet based on blood type (A, B, O, AB) will result in weight loss and overall health.	Blood Type O: 1000	44	29	27	Weight maintenanceOverall Health
		Blood Type A: 1150	55	10	34
		Blood Type B: 1200	55	22	23
		Blood Type AB: 1200	56	25	20
Body for LifeBill Phillips, Michael D’OrsoWilliam Morrow, 1999	This book focuses primarily on exercise, and recommends 6 small meals per day for 6 weeks, consisting of lean meats, vegetables, whole grains, healthy fats, and fish in addition to strenuous exercise.	1270	45	45	10	Exercise and nutrition for quick weight loss
The New Cabbage Soup DietMargaret DanbrotSt. Martin's Paperbacks, 2004	Very-low calorie diet plan, based on the theory that monotony will cause the person to stop eating. Only food consumed is cabbage soup supplemented occasionally by specific fruits and vegetables	700	57	18	25	Weight Loss
The Cheaters DietPaul Rivas, MDHCI, 2005	Based on the plate method: 1/2 plate vegetables, 1/4 whole grains, 1/4 lean protein. Dr. Rivas claims that you must cheat on the weekends to “stroke your metabolism and boost fat loss.” He suggests eating “whatever you want” from 9am on Saturday to 9pm on Sunday.	1200-excessive calories	50	20	30	Weight Maintenance
The Diet Solutionhttp://www.thedietsolutionprogram.com/N/A	Diet that promotes organic and “natural” foods, free of processing, regardless of the macronutrient composition. Excludes soy products.	928	34	20	46	Weight loss
Eat This, Not That!David Zinczenko with Matt GouldingRodale Books, 2009	Written by the editor-in-chief of Men’s Health magazine, aimed at male readers who eat mainly at fast food restaurants. Attempts to provide readers with alternative options to calorie laden fast food choices.	-	-	-	-	Weight maintenance
Flat Belly DietLiz Vaccariello and Cynthia SassRodale Books, 2009	Premise is to trim calories to 1600, add a mono-unsaturated fatty acid at every meal, eat every four hours, and to perform regular exercise to lose weight and belly fat.	1600	42	25	33	Weight loss
French Women Don't Get FatMireille GuilianoVintage, 2007	Lifestyle changes illustrated through an autobiography of the author	1200-1300	43	22	45	Low Calorie
Change Your Genetic Destiny (The GenoType Diet)Dr. Peter J. D’Adamo with Catherine WhitneyBroadway, 2009	An expansion on the concept of the Blood Type Diet, created by naturopathic physician Dr. D’Adamo. Dr. D’Adamo identifies the six “GenoTypes”: the Hunter, the Gatherer, the Teacher, the Explorer, the Warrior, and the Nomad. Diet helps consumers map out their genetic makeup and discover which “GenoType” they are.The theory is that readers can reprogram their gene responses to lose and maintain weight, among other health improvements, by choosing foods that enhance each GenoType, and avoiding foods that do not.	-	-	-	-	Weight maintenanceOverall health
The New Glucose Revolution: The Authoritative Guide to the Glycemic IndexJennie Brand-Miller, Phd; Thomas Wolever, MD, Phd; Kaye Foster-Powell; Stephen Colagiuri, MDMarlowe and Co., 2006	The theory is that simple carbohydrates cause spikes in blood sugar levels, causing recurrent hunger. Recommends eating low glycemic-index foods (i.e., whole grains, protein) to stave off hunger and weight gain.	1200	55	24	21	Weight Maintenance
LearnKelly BrownellAmerican Health Publishing Company, 2004	Lifestyle, exercise, attitudes, relationships and nutrition following government recommendations.	1650	55	15	30	Weight Maintenance
Mediterranean	Recommends grains, vegetables, and sources of healthy fats (e.g., olive oil and nuts).	-	45	20	35	Weight Maintenance
The Perricone PrescriptionDr. Nicholas PerriconeHarper Paperbacks, 2004	Anti-inflammatory foods eaten to reverse aging. Unlimited salmon.	1300	35	39	26	Anti-inflammatory/Overall Health
Pritikin Program for Diet and ExerciseNathan PritikinBantam, 1984	Six meals per day, no portion control.	-	80	10	<10	Low fatVegetarianWeight loss
The Complete Scarsdale Medical DietDr. Herman TarnowerBantam, 1982	Artificial sweeteners and appetite suppressants are recommended.	1000	21	46	35	Weight loss
Sensa Weight-Loss Program (The Sprinkle Diet)Dr. Alan Hirsch, MD, FACPHilton Publishing, 2009	Flavorless sprinkles (“Tastanants”) sprinkled on food, helps dieters eat less and feel full faster.	-	-	-	-	Weight loss
The Serotonin Power DietJudith J. Wurtman, PhDNina T. Frusztajer, MDRodale Books, 2006	The authors claim carbohydrate-rich snack eating will decrease stress and help dieters lose weight by producing more serotonin.	1500	62%	18%	20%	Weight loss
The Sonoma DietConnie Guttersen RD, PhDMeredith Books, 2005	Influenced by a Mediterranean plant-based diet. This three phase diet places emphasis on a variety of flavorful, nutrient dense "power foods" such as almonds, bell peppers, blueberries, broccoli, grapes, olive oil, spinach, strawberries, tomatoes, and whole grains.	1500 for men	50-55	15-20	30	Low Calorie
The Sonoma DietConnie Guttersen RD, PhDMeredith Books, 2005		1200 for women	50-55	15-20	30	Low Calorie
South Beach DietDr. Arthur AgatstonRodale, Inc., 2003	3 phase book by Arthur Agaston MD; Low carbohydrate*Phases based on a 140-pound, 40-year-old, lightly active woman	Phase 1: 1850	16	38	46	Low Calorie
		Phase 2: 1450	37	26	40	Low Calorie
		Phase 3: 1750	31	29	40	Low Calorie
The Spectrum DietDr. Dean Ornish, MDBallantine Books, 2008	A lifestyle change; find where you fall on Ornish’s food “spectrum” (Group 1 being the healthiest, Group 5, the least healthy) and make changes according to your desired health outcomes (e.g., weight loss, weight maintenance, reduced risk of cancer, etc.).Plan calls for regular exercise (aerobic, resistance training, and flexibility), stress management (yoga, meditation), nutrition advice (low-fat, vegetarian), and nurturing relationships.	1580	70	20	10	Low-fat/vegetarian for overall improved health
Sugar BustersH. Leighton Steward; Morrison C. Bethea, MD; Sam S. Andrews, MD; Luis A. Balart, MDBallantine Books, 1999	Cut or completely eliminate dietary sugar to trim fat	1200	40	30	30	Weight loss
The Supermarket DietJanis Jibrin MS, RDHearst, 2007	Provides shopping lists, meal plans, recipes and snacks. The book begins with at two week boot camp phase. The author helps readers select which calorie level is the best fit for them, and how to troubleshoot problems if the calorie level does not seem to be yielding results.	1200-1500	50	20	30	Low CalorieWeight Maintenance
The Ultimate Weight SolutionDr. Phil McGrawFree Press, 2004	Dr. Phil McGraw authored this 3 phase diet book	Phase 1: 1300	47	36	17	Low Calorie
		Phase 2: 1100	49	32	19	Very low calorie
		Phase 3: 1820	52	27	17	Weight Maintenance
The Volumetrics Weight-Control PlanBarbara Rolls, PhDHarperTorch, 2002	Focuses on satiety and feeling full, by filling up on high volume foods with low energy density (e.g., soup)	1700	61	23	18	Weight lossWeight maintenance
YOU: On a Diet (Revised Edition)Dr. Mehmet Oz and Dr. Michael RoizenFree Press, 2009	Weight loss with an emphasis on waist measurement and its relationship to health.	1700	46	21	33	Weight lossWight maintenance
The Zone DietDr. Barry SearsThorsons, 1999	Balances carbohydrates, protein, and fat to stabilize the hormones that trigger hunger and weight gain.	1700	40	30	30	Weight loss

10.5 Web-Based Treatment Programs and Resources

The Internet provides some excellent resources for those who want and need more information. However, it also includes sites with questionable recommendations, so individuals should proceed with caution. Web-based resources are discussed below.

10.6.1 Available Programs

Two types of programs are available. First are those that primarily provide information. Second are those that counsel the individual and provide low calorie diets and other advice. Sound Internet resources that can help those who are trying to control their weights are listed in Table 25. These fall into the category of informational resources. The reader needs to be aware that not all sites providing advice and information are sound. It is best to trust the sites sponsored by government, professional, and voluntary associations with some standing and expertise in the weight control field. A new entry into the weight loss arena in recent years is the web-based weight control program (210). These are examples of the second type of program. Resources include chat rooms, diet and exercise information, and often products that are for sale. For example, Nutrisystem®.com requires the purchase of prepackaged foods in order to access their web site. The ediets.com website, another weight control program, charges a monthly fee to use its site. It provides shopping lists from which consumers self-select foods, and also it provides general advice.

These web-based commercial offerings vary in their quality, some are very good and others are quite poor.

Table 25. Internet Resources for Weight Control (164-167)
Type of Site and Name	Internet address and Comments
Advice and Information on nutrition and weight control
American Dietetic Association	www.eatright.orgThis is the website for nutrition professionals. Membership allows entrance to the Journal of the American Dietetic Association and the Evidenced Based Library.
Shape Up! America	www.shapeup.orgProvides good yet minimal nutrition education information for patients. Helpful sample menus are provided for 1500 and 2000 calorie diets as well as practical ways to increase physical activity.Founded in 1994, Shape Up America! is a 501(c)3 not-for-profit organization committed to raising awareness of obesity as a health issue and to providing responsible information on healthy weight management.
American Obesity Association	www.obesity.orgComprehensive website dedicated to obesity. Provides helpful information for patients as well as links to other nutrition/fitness resources. This website also offers information on treatment, prevention, education, various aspects of public policy, and obesity research for professionals.
National Institutes of Health	www.nutrition.govExtremely helpful resource for patients and health professionals alike. The website is home to the Dietary Guidelines, Mypyramid which is an interactive diet and physical activity planner, and an abundance of information regarding health and nutrition.
Weight-Control Information Network (WIN)	http://win.niddk.nih.gov/A helpful information service of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) for health professionals and the public. WIN was established in 1994 to provide the general public, health professionals, the media, and Congress with up-to-date, science-based information on obesity, weight control, physical activity, and related nutritional issues.
Web-Based Help for Dieters
Cyberdiet.com	www.cyberdiet.comThe program is $38.87 per 3 months or $77 annually via www.dietwatch.com. Resources on fitness, nutrition, motivation, wellness, recipes, and emotional health are included in each counseling plan. RDs provide the nutrition recommendations and accredited health professionals provide other health counseling/information.
WebMD Nutrition Resources	http://www.webmd.com/diet/default.htmThis is an extremely useful resource for patients. The site provides a BMI calculator, calorie counter, diet evaluator, fitness and diet journal, a large food/nutrient database, a fiber calculator, helpful articles, videos, and slideshows on eating healthfully, and charts that can be personalized.
Ediets.com	www.ediets.comProvides a Pyramid based reducing diet and food list, fitness information, healthy recipes, social networking community, and charts and dieting tools. Cost is currently $17.95 for the first 4 weeks. Meal delivery options are also available for $19.95 per day. This option includes 3 meals per day and a snack or dessert in addition to a social networking community and nutrition and fitness guidance.
Weight Watchers® Online	www.weightwatchers.comSelf-help version of Weight Watchers® program provides a diet plan and fitness information with exercises. Cost is currently $53.85 per month for 3 months and $17.95 for each month after. Very consumer friendly.
Lifepractice.com	www.lifepractice.comThe Life Practice program deals with exercise, nutrition, sleep habits and stress management. A personal coach is assigned to each member. Frequent e-mails and daily tracking of the member's progress is standard. The cost is $3.50 per week.
Fitday.com	http://fitday.com/This website is a free online journal that tracks and analyzes food intake, exercise, and weight loss goals.
caloriescount.com	www.caloriescount.comThis site boasts several online calculators and tools that help the participant keep track of their weight loss, such as the exercise/calories burned calculator and online diet meal plans given the participants appropriate caloric range, which may also be determined from the site. These tools are free of charge. Access.
SparkPeople®	www.sparkpeople.comA comprehensive website with free nutrition, health, and fitness tools, support, and resources. This website boasts a large online social support network and may particularly help those with a preference towards online community networks.
Diet.com	www.diet.comA nutrition and health website with tools to aid in weight loss, healthy living, and wellness. Basic membership is free and includes access to online support groups, articles, and useful tracking tools. However, premium membership, including all of the above in conjunction with a customized diet plan, personalized exercise plan and coping plan, personalized expert advice, and individualized weekly self checklists to help keep you on track, requires a $19.95 initiation fee and depending on the plan may range from $9.95 to $19.95 per month.

10.6.2 Candidates

Those who are overweight or moderately obese with few risk factors, and who need additional support and information after they have been screened by a physician on weight reduction, may find these resources useful. They are not freestanding and need to be administered in conjunction with some additional health and dietary counseling about a hypocaloric diet from a physician or registered dietitian.

10.6.3 Advantages

The Internet is widely available at all times of the day or night, at low cost. For example, the US Army has developed a web-based dietary advice program that can be used at Army bases around the world.

10.6.4 Disadvantages

High-risk patients, especially those who lack economic resources, may attempt to use these Internet sites for the primary treatment of their condition. Also, some sites provide inappropriate or wrong advice and there is little personal supervision or support of the dieter. Peer support networks and chat rooms can be beneficial to a patient trying to find support for weight loss, but there is also a risk that peers may provide incorrect advise or promote unsubstantiated weight loss techniques.

10.6.5 Safety and Effectiveness

The safety and effectiveness of internet sites for weight reduction has not been established (105). Only recommended sites should be trusted.

10.7 Voluntary Self-Help Programs

Self-help programs led by laypersons are voluntary programs that charge very low or no fees. National organizations include TOPS Club, Inc.® (Take Off Pounds Sensibly), OA (Overeaters Anonymous®), and others. These programs are designed primarily to provide group support to those who have weight problems, rather than to provide and supervise weight reduction diets.

10.8 Mobile Applications for Weight Loss

As people’s lives become more mobile, so do the tools that help them lose weight. Calorie tracking programs are available to monitor daily intake, and fitness programs provide sample workouts and activity logs. Other apps are available to promote water consumption and provide tips and support. As with web-based resources, these should be used in conjunction with physician or dietician-led interventions, and not as a replacement for them. Though research on the efficacy of these programs is still scant, one recent study suggests that combining some form of counseling alongside a smart-phone based platform led to greater results than either counseling or smart-phone use alone (237). Examples of current available apps and the platforms on which they are available are summarized in Table 26.

App Name	Cost	Available Platforms	Features
Table 26. Mobile Apps to Aid in Weight Loss
My Fitness Pal	free	Apple, Android, Windows, Blackberry, and online (http://www.myfitnesspal.com)	Features include a calorie tracker, activity tracker, weight tracker, body measurement tracker, water tracker, extensive food list including restaurants, barcode scanner, breakdown of intake including protein, fat, carbohydrates, fiber, and vitamins and minerals, daily and weekly summaries, ability to add friends, and compatibility with fitbit, map my fitness, and other apps and devices.
Lose It!	Free (premium upgrade for $39.99/yr)	Apple, Android, and online (https://www.loseit.com)	Features include calorie tracker, activity tracker, weight tracker, barcode scanner, extensive food list with restaurant options, ability to add recipes, breakdown of intake by protein, fat, and carbohydrates, daily and weekly summaries by email, ability to add friends, compatibility with Nike+ fuelband. Premium features include additional compatibility with apps and devices, hydration and sleep trackers, body measurement tracker, and blood glucose and blood pressure trackers.
Mapmyfitness	free (premium upgrade for $29.99/yr for apple and online)	Apple, Android, Blackberry (limited), and online (http://www.mapmyfitness.com)	Features include allowing users to track a variety of activities in real time and after the fact, search for and save routes for walking, jogging, biking, and hiking, a food tracker, the ability to play music from music library on phone, add friends and share workouts. “Mapmyrun” and “Mapmyride” are related apps for running and biking respectively. Premium features include training plans, training features, and live location tracking.
Hy	free	Apple	Features include allowing users to set water goals in ml or fl oz, add water consumed in increments of 50 or 100 ml (2 or 4 fl oz), the ability to set reminders to consume water, and facts about the importance of water consumption displayed daily.
EaTipster	free	Apple	Features include daily tips on healthy eating from dietitians, ability to save and share tips, and the ability to set reminder for tips

11. Summary of Weight Loss Phase

Current guidelines for the composition of weight reducing diets, as discussed above, are outlined in the 2010 Dietary Guidelines for Americans (Table 1), as well as the MyPlate.gov website.

12. Weight Maintenance Phase of Weight Control

Once obese individuals have lost weight, their healthier weight and current fat mass must be maintained. This is the weight maintenance phase of weight control. It involves alterations in dietary intake and physical activity from levels that are different to those at the onset of dietary treatment. Energy needs are lower to stay in energy balance than they were prior to weight reduction, even though weight was lost. This is because both fat and lean body mass is lost during the weight reduction phase. With a loss of lean body mass comes a decrease in metabolically active tissue, which then reduces resting metabolism. In addition, it takes less effort for an individual to move with a now lighter body, so the energy cost of physical activity is reduced. The implications are that a slight decrease in energy intake from prior levels and an increase in energy output is necessary during weight maintenance. There is a need for continued attention to these factors by both the physician and patient. Behavior modification is necessary to sustain lifestyle changes developed during the weight reduction phase. It is best initiated during the weight loss phase, and maintained thereafter. All too often the weight maintenance phase is neglected or ignored, and weight is regained over the long term (71). Some factors that seem to be associated with long-term successful weight maintenance include continued regular exercise and to a lesser extent, use of low calorie, low fat diets relatively high in fruits and vegetables. Also, continued self-monitoring of the amount and type of food consumed and of physical activity levels may help (71).

12.1 Nutrient Needs

Although energy needs are less during weight maintenance, the requirements for protein, essential fatty acids, carbohydrate, dietary fiber, vitamins and minerals are similar to those of any normal adult. The 2010 Dietary Guidelines for Americans recommend that all Americans, including those who are watching their weight, to adopt the habits listed (see Table 1).

There is currently much debate about the ideal macronutrient distribution in diets for weight maintenance, but at present very few long-term studies are available on the effects of macronutrient content on weight maintenance. A low-fat, reduced-energy is the best studied diet and the most prescribed for weight maintenance (AND).

12.1.2 Carbohydrate

A recent study of the self-selected diets of free-living American adults found that diets high in carbohydrates (above 55% of calories) were lower in total energy and were associated with lower BMI’s than those consuming fewer carbohydrates. The nutrient density (amount of the nutrient per calorie consumed) of those with higher carbohydrate intakes was also higher for vitamins A, Vitamin C, carotene, folate, calcium, magnesium, and iron, but lower in vitamin B-12 and zinc than those with a lower intake of carbohydrates. Also, the high carbohydrate group ate more low-fat foods, grain products and fruits in addition to lower sodium intakes (214). Although individuals who choose to go on a very-low-carbohydrate diet may see increased weight loss within the first six months, these results are not sustained at 12 months (241). It is thought that this diet is not sustainable for long-term weight maintenance and instead, the diet should be a slight reduction in energy all together as well as an increased focus on the reduction of fat (241).

12.1.3 Energy Density

Other studies suggest that energy density of the diet rather that the macronutrient composition of the diet affects energy intake the most (215;216). One review found that low fat, high fiber diets were the most effective in promising weight loss, and that their effects appeared to be associated with energy density (217). Whether this is true in weight maintenance remains to be determined. There may be macronutrient effects on hunger and satiety that operate through endocrine and metabolic mechanisms such as leptin, insulin, grhelin, adiponectin and other hormones, which are only now being discovered. These hormones regulate food intake and may be altered. Additionally, the macronutrient composition of habitual diets also affects health risks. Finally, psychological and behavioral factors may make different macronutrient combinations more acceptable to some people. Currently, these topics are the subject of much debate, but research is needed to clarify what and which nutrient composition is optimal. What is currently recommended by the Academy of Nutrition and Dietetics with a strong rating is that an individualized reduced calorie diet is imperative for weight maintenance. By reducing fat, an individual is able to cut out more calories but it is suggested that both fat and carbohydrate be decreased (241).

Other forms of low energy dense diets would be the use of meal replacements or very-low-energy diets. Using meal replacements can be helpful for those who have trouble planning and preparing meals. They can also be helpful for those who experience a large amount of anxiety during meal times. An individual is able to replace one or two meals or snacks with these meal replacements with known nutrient content to help them stay at a calorie deficit of 500 – 1000 cal/day (241). This is something that could be used periodically to help patients get back on track after a relapse of poor food choices and is something that can be maintained long-term. Very-low-energy diets use meal replacement bars or shakes as the sole source of energy during the weight loss phase, however, this is not suitable for weight maintenance and instead used for quick weight loss and requires additional medical monitoring (241).

12.1.3 Fat

Although much remains to be discovered about the optimal dietary pattern for weight maintenance, a strong case can be made for keeping dietary fat levels below 30% of calories. In studies in which dietary fat was reduced from 35 to 25% of calories with no other recommendations, energy intake was reduced and weight was lost (218). It was estimated that reducing fat by 10% to within the range of 20 to 30% of calories would result in a loss of about 16 grams of body fat a day as a result of reduced energy intake. However, moderation in caloric intake is also necessary. Studies of free-living humans ranging from dietary changes produced only modest body weight losses of about one to three kilograms (182;219;220). Low fat diets consumed on an ad-libitum basis tend to be high in carbohydrate, but LDL cholesterol decreases, plasma triglycerides tend to normalize, and so do HDL/total cholesterol ratios (221). Finally, weight control may be easier (222).

12.1.4 Dietary Fiber

Although the influence of dietary fiber on energy regulation is still not clear, there is evidence that increased dietary fiber intake of about 15 grams appears to be associated with decreased energy intake and body weight of about two kilograms over several months. These effects may even be greater in overweight persons (181). However, these effects are not yet confirmed. Since dietary fiber intake is currently low, only about 15 grams per day in most Americans, and recommendations are for nearly twice that much, increased fiber levels seem to be appropriate, regardless.

12.1.5 Vitamins and Minerals

It is important to meet the current dietary recommendations of 3 – 4 servings of low-fat or non-fat dairy in order to get the daily suggested values of calcium and vitamin D. Research suggests that those with lower calcium intake have increased body weight (241). However, the mechanism by which this works is still unclear.

12.1.6 Lessons from Long Term Maintainers: Importance of Increased Physical Activity

Long-term follow-up of health outcomes demonstrates the need for permanent changes in weight toward healthier levels. The relative lack of effects of temporary downward fluctuations underscores the need for long-term weight maintenance. Data collected from individuals successful at weight loss and maintenance have enhanced our understanding of the most effective strategies in the long-term maintenance of healthier weights and prevention of relapse. Attention to moderation in dietary intake and the maintenance of high levels of physical activity is vital (223-226). Behavioral and attitude adjustments are also important. Encouraging data suggest that behaviors associated with maintenance of weight loss require less effort and become more pleasurable over time (29). In a recent telephone survey, 48% of individuals who had ever lost more than 10% of their body weight had maintained this loss for at least one year, and 26% had maintained for at least 5 years (227). Although these data are self-reported, they suggest progress in the avoidance of relapse and weight gain.

13. Adopt a Long-Term Eating Pattern to Maintain Weight: Adequate Nutrients within Calorie Needs

Most individuals appear to be aware of and use recommended measures, such as increased physical activity, decreased fat intake, decreased food portions, and decreased energy intakes. The problem is that they do so, but not for enough of the time. However, it is also true that dieting efforts often fail, and weight is often rapidly regained, probably negating predicted health benefits. Chronic dieters tend to be food-preoccupied, distractible, emotional, binge-prone, and unhappy, particularly when the diets are very restrictive (228). It is thus important to foster a healthy, balanced, stable relationship with food and diet. Health professionals can play a vital role in helping patients develop such a relationship.

14. Conclusions: Is Dieting Worth It?

About 39% of women and 21% of men in Western countries have ever tried to lose weight, and approximately 24% of women and 8% of men report that they currently are on a “diet”. In contrast, about 25% of men and 30% of women report that they are watching what they eat to avoid weight gain or to maintain their weights at current levels (229). Hypocaloric diets to induce loss of body fat therefore appear to be a common component of the weight control efforts of many people.
These realities and disadvantages have rightly led to questions about whether dieting is “worth it”, and whether the treatment is worse than the disease. Certainly they suggest that quality of life measures should be included in studies of reducing diets.
This chapter has stressed the role of the dietary treatment of obesity as a part of a comprehensive program of weight control that includes increased physical activity, lifestyle modification, appropriate intakes of nutrients to minimize chronic disease risk, and eating patterns that maximize quality of life. Such dietary treatment in those with mild to moderate obesity helps to decrease risk factors relative to baseline weights after five years. Therefore some health benefit, although it is limited, may be present. However, the health risk/benefit may be negative when dieting entails a cycle of rapid loss followed by equally rapid weight gain. From the standpoint of quality of life and mental health, psychosocial problems do not appear to be inevitable accompaniments of weight loss (184). Therefore, on balance, dietary approaches to obesity management do appear to be worthwhile, if and only if they are viewed as only one component of a long-term weight control program to keep weights and risks at healthier levels. Weight control is “Worth it”!

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ABSTRACT

INTRODUCTION

IODINE AVAILABILITY AND TRANSPORT

UPTAKE OF IODINE BY THE THYROID

IODINE IN OTHER TISSUES

THYROPEROXIDASE (TPO)

H 2 O 2 GENERATING SYSTEM

THYROGLOBULIN (Tg)

THYROGLOBULIN IODINATION AND HORMONE SYNTHESIS

HORMONE STORAGE

THYROGLOBULIN ENDOCYTOSIS

PROTEOLYTIC CLEAVAGE OF THYROGLOBULIN

CONTROL OF HORMONE SYNTHESIS

Reference List

Abstract

Key messages

INTRODUCTION

ANORCHIA

Congenital Anorchia

Acquired Anorchia

LEYDIG CELL HYPOPLASIA

Spermatogenic failure

GERM CELL APLASIA (SERTOLI CELL ONLY SYNDROME)

SPERMATOGENIC ARREST

HYPOSPERMATOGENESIS

Mixed Atrophy

NUMERICAL CHROMOSOME ABERRATIONS

Klinefelter Syndrome

XX-Male Syndrome

XYY-Syndrome

STRUCTURAL CHROMOSOME ABERRATIONS

Structural Aberrations of the Autosomes

Structural Aberrations of Sex Chromosomes

Y CHROMOSOME MICRODELETIONS

OUTLOOK: NEW TECHNOLOGIES, NOVEL GENETIC CAUSES

REFERENCES

ABSTRACT

Introduction

NORMAL PUBERTY FOR GIRLS (IT’S OCCURRING EARLIER!): A basis for the Definition of Abnormal Puberty

Onset of Normal Pubertal Landmarks

Determinants of Normal Pubertal Growth

Genetic Influences of Growth

Hormonal Determinants of Growth (Some gene mediated)

DEFINITION OF ABNORMAL PUBERTY

PRECOCIOUS PUBERTY

Overview

Central Precocious Puberty

Contemporary Issues for Management of CPP

Iatrogenic sexual precocity

Premature Thelarche

Premature Menarche

Contrasexual precocity

Virilizing precocious puberty in girls and isolated precocious adrenarche

DELAYED PUBERTY

An Overview of Delays within the H-P-O Circuit (Delays of Secondary Sexual Development and Menarche)

DISORDERS IDENTIFIED IN PATIENTS WITH EITHER DELAYED PUBERTY OR MENARCHE

Hypogonadism

Hypergonadotropic Hypogonadism

Turner Syndrome

Normal Chromosomes

Molecular Findings

46,XX Gonadal Dysgenesis.

46,XY Gonadal Dysgenesis.

Contemporary Issues for Management

Hypogonadotropic Hypogonadism

Constitutional delay

Acquired Abnormalities

Congenital Abnormalities

Molecular Findings

Contemporary Issues for Management

Eugonadism

Anatomic Abnormalities

Molecular Findings

Contemporary Issues for Management

CHRONIC ANOVULATION

DISORDERS OF SEXUAL DEVELOPMENT

Molecular Findings

Contemporary Issues for Management

REFERENCES

Abstract

H ₂ O ₂ GENERATING SYSTEM