Metabolic Aspects of PCOS: Treatment With Insulin Sensitizers 2015 Ed.

6. PCOS Therapy

Mariagrazia Stracquadanio¹ and Lilliana Ciotta¹

(1)

Obstetrics and Gynecological Pathology, P.O. “S. Bambino”, University of Catania, Catania, Italy

Keywords

DietExerciseMetforminInositolStatinsAntioxidantsGlucomannan

Gynecologists usually treat PCOS only as an endocrine disorder, without recognition of the very important part that insulin resistance plays in the syndrome.

In this section, the way to treat PCOS from a metabolic point of view, without dwelling on the use of oral contraceptives and antiandrogen drugs, will be discussed.

Lifelong strategies that improve the care of women with PCOS are essential, because of the chronic nature of the syndrome and the young age at which all the symptoms begin to manifest [1].

A valid therapeutic protocol for PCOS includes diet, physical exercise, and insulin-sensitizing agents such as metformin and inositol.

For example, in fact, a normal BMI is associated with a positive fertility outcome, and fertility specialists recommend achieving this BMI before IVF (in vitro fertilization): in fact, these techniques are invasive and expensive and have low success rates, so it seems logical to improve BMI and to support hormonal balance through diet, exercise, and nutrition supplements [2].

6.1 Diet and Exercise

As explained previously, a few evolutionary biologists suppose that many genetic hormonal tendencies contributing to PCOS have their origin in the switch from the pre-agrarian age diet to the current diet. The rapidly increasing rates of diabetes, heart disease, and PCOS coincide with the rapid changes in the modern human diet [2].

All women suffering from PCOS benefit from dietary therapy and exercise; in fact, dietary and lifestyle interventions are considered among the first-line treatments for PCOS.

There is no PCOS diet that will reverse the syndrome, but there are several dietary principles that a patient should follow to improve the symptoms.

Weight reduction leads to improvements of insulin sensitivity [3] and lipid profile [4]; it ameliorates hyperandrogenism (SHBG increase, FAI and testosterone decrease) and menstrual cycle rhythm [4–6], with reductions in adiposity from the truncal–abdominal area [5]. Moreover, there is evidence that these changes exert important beneficial effects also in the longer term on disorders such as type II diabetes mellitus, cardiovascular disease, and certain cancers (endometrial, breast, and colon cancer), compared with weight loss alone [7–9].

In most of the dietary studies in women with PCOS, improvements in metabolic and reproductive outcomes have been closely related to improvements in insulin sensitivity, suggesting that dietary modification (qualitative and quantitative) designed to improve insulin resistance may produce greater benefits than those achieved by energy restriction alone [7].

Clinicians prescribing lifestyle modifications must consider the patient’s capacity to sustain diet and exercise adherence and weight maintenance over time for the clinical benefits on PCOS to continue.

Considering how difficult it is for many patients to change their lifestyle, pharmaceutical modification of weight control could be an additional necessary therapeutic tool, such as the lipase inhibitor orlistat [10].

In some studies on overweight and obese women with PCOS, the use of orlistat has demonstrated an improvement in both metabolic and hormonal parameters [11, 12].

Orlistat is an antiobesity drug with minimal systemic absorption, and therefore, any effect of this drug is a result of weight loss and not the direct effect on ovaries.

The proposal therapeutic dose is 120 mg three times daily, before each meal, for 3 months, during which the patient must be able to lose at least 5 % of its total weight.

6.1.1 PCOS Dietary Recommendations

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Data in literature show that a diet with 50 % of total calories from carbohydrates (with a low glycemic index), 30 % from fat (mostly mono- and polyunsaturated fat, less than 10 % from saturated fat), 20 % from proteins, and high in fiber is the most appropriate for patients with PCOS [32].

The optimal frequency of food intake has yet to be determined: however, a regular pattern with low intake from snacks is advisable [8], and high-calorie intake at breakfast with reduced intake at dinner is suggested, because it leads to reduced overall insulin levels [33–35].

6.1.2 Glycemic Index (GI)

It has been shown that eating foods with a low GI improves glucose control in women with PCOS and diabetes.

The glycemic index indicates the rate in which glycemia increases after taking a quantity of “X” food containing 50 g of carbohydrates.

Foods with carbohydrates that break down quickly during digestion and release glucose rapidly into the bloodstream tend to have a high GI; foods with carbohydrates that break down more slowly, emitting glucose more gradually into the bloodstream, tend to have a low GI [2].

The concept was developed by Dr. David J. Jenkins and colleagues [36] in 1980–1981 at the University of Toronto in their research to find out which foods were best for people with diabetes. A lower glycemic index suggests slower rates of digestion and absorption of the foods’ carbohydrates and may also indicate greater extraction from the liver and periphery of the products of carbohydrate digestion.

A lower glycemic response usually relates to a lower insulin demand but not always and may improve long-term blood glucose control and blood lipids [37]. The glycemic index of a food is defined as the incremental area under the 2-h blood glucose response curve (AUC) following a 12-h fast and ingestion of a food with a certain quantity of available carbohydrate (usually 50 g). The AUC of the test food is divided by the AUC of the standard (either glucose or white bread, giving two different definitions) and multiplied by 100. The average GI value is calculated from data collected in ten human subjects. Both the standard and test food must contain an equal amount of available carbohydrate. The result gives a relative ranking for each tested food [38]. The GI Symbol Program is an independent worldwide GI certification program that helps consumers identify low-GI foods and drinks. The symbol is only on foods or beverages that have had their GI values tested according to the standard and meet the GI Foundation’s certification criteria as a healthy choice within their food group. GI cutoffs are listed in Table 6.1.

Table 6.1

Glycemic index cutoffs

Glycemic index cutoffs
High	≥70
Moderate	50–70
Low	<50

Of course, the glycemic index has also its limitations: the index calculations are not accurate because the behavior of foods in different individuals can change, and judging the diet by GI alone does not give the whole portrait of the diet [2].

Moreover, GI values depend on how foods are cooked: cooked carrots have a higher GI than raw carrots because cooking breaks down the fiber and the glucose can be absorbed much more quickly. Cooking with a bit of salt or vinegar may lower the GI of many vegetables because this causes many molecules, not just the sugars, to be broken down, which results in trapping some of the starches in complex structures that are digested more slowly [2].

Furthermore, for some people, a food consumed in the morning on an empty stomach will spike the blood sugar more than the same food eaten later in the day after having breakfast: patients with good blood sugar control in general will show less of a spike in blood sugar than someone with poor blood sugar control [2].

PCOS women should follow some useful GI advices for their daily diet:

· Eat five to ten different whole fresh fruits, vegetables, and legumes each day.

· Avoid a diet that consists predominantly of the food highest on the glycemic index.

· Substitute foods high on the GI with foods lower on the GI: for example, eat boiled green beans (GI of 15) instead of boiled potatoes (GI of 100) with dinner (Table 6.2).

Table 6.2

Foods’ glycemic index list

Foods’ glycemic index
Sweeteners
Corn syrup	100
Table sugar (sucrose)	100
Rice syrup	65
Honey	54
Fructose	10
Stevia	0
Grains
White rice	90
Rice cakes	84
Wild rice	81
Corn chips	72
Cornmeal	70
Couscous	65
Brown rice	55
Pop corn	55
Whole wheat	48
Whole amaranth	35
Bread
Rice bread	100
Polenta	98
Baguettes	95
Doughnuts	76
Croissant	70
White bread	70
Pancakes	67
Kamut bread	54
Rye bread	50
Pasta, whole grain	44
Wheat germ	15
Cereals
Instant oats	92
Puffed rice	85
Grape-Nuts	67
Oat bran	15
Nuts and seeds
Chestnuts	60
Peanut butter	40
Sesame seeds	35
Almonds	15
Hazelnuts	15
Pistachios	14
Walnuts	14
Peanuts	14
Legumes and beans
Fava beans	50
Black beans	35
Hummus	35
White beans	35
Lentils	29
Soybeans	18
Green beans	15
Tofu	14
Vegetables
Potatoes, baked	100
Potatoes, boiled	84
Carrots, cooked	80
Beets, cooked	64
Corn	55
Peas	44
Coconut	35
Tomato sauce	35
Carrots, raw	30
Asparagus	15
Cucumbers	15
Lettuce	15
Mushrooms	15
Olives	15
Spinach	15
Tomatoes	15
Zucchini	15
Avocados	10
Fruits
Watermelons	90
Pineapples	66
Apricots	57
Strawberries	56
Mangos	55
Bananas	52
Grapes	50
Oranges	46
Apples	39
Peaches	30
Raspberries	25
Cherries	25
Others
Beer	110
Rice milk	84
Mango juice	55
Orange juice	45
Coconut milk	40
Soy milk	36
Yogurt, low-fat fruit	33
Almond milk	30
Dark chocolate	25
Lemon juice	20
Pesto	15
Vinegar	5
Water	0

· Increase fiber intake: fiber helps to slow the digestion of carbohydrates and improves insulin resistance. If a food high on the GI is loved, patient should take care not to consume it often and aim to eat only a small portion of it combined with high-fiber foods that reduce the glycemic index [24, 25].

· Eat legumes to lower the high-GI foods in the meals: legumes are low on the GI and contain an impressive amount of fiber and good-quality protein, which can serve to blunt the glycemic load. Moreover, legumes contain pinitol, a relative of D-chiro-inositol, noted for improving insulin resistance [2].

· Avoid overeating foods high on the glycemic index. The GI of a food can be tempered by the quantity consumed. For example, a piece of candy might have a very high glycemic index, but eating just one little piece will not result in a high glycemic load on the body; if the patient eats two pieces of white toast, jam, brown potatoes, and a sugar- or corn syrup-sweetened fruit drink for breakfast, she is putting a high glycemic load on her body, and the blood sugar will remain high for several hours as her body works to process the large amount of high-GI foods [2].

· Evaluate the whole meat, rather than individual food items, to make sure the patient is preparing meals that will not spike her blood sugars.

6.1.3 Glycemic Load (GL)

Some authors believe that the glycemic load (GL) is a more useful measure of food value than the glycemic index alone.

Glycemic load accounts for how much carbohydrate is in the food and how much each gram of carbohydrate in the food raises blood glucose levels.

GL is a GI-weighted measure of carbohydrate content [39].

For instance, watermelon has a high GI, but a typical portion of watermelon does not contain many carbohydrates, so the glycemic load of eating it is low. Whereas glycemic index is defined for each type of food, glycemic load can be calculated for any size serving of a food, an entire meal, or an entire day’s meals.

GL cutoffs are listed in Table 6.3.

Table 6.3

Glycemic load cutoffs

Glycemic load cutoffs
High	≥20
Intermediate	11–19
Low	<10

Foods that have a low GL in a typical serving size have usually a low GI. Foods with an intermediate or high GL in a typical serving size range from a very low to very high GI (Table 6.4).

Table 6.4

Foods’ glycemic load list

Glycemic load (per 100 g serving)
Baguette	15
Banana	16
Potato	20
Carrots	2
Rice	30
Watermelon	4

For detailed information about all the foods, visit the website www.​glycemicindex.​com

6.1.4 Insulin Index

The insulin index is a measure used to quantify the typical insulin response to various foods. The index is similar to the glycemic index and glycemic load, but rather than relying on glycemia levels, the insulin index is based upon insulinemia. This measure can be more useful than either the glycemic index or the glycemic load because certain foods (e.g., lean meats and proteins) cause an insulin response despite there being no carbohydrates present, and some foods cause a disproportionate insulin response relative to their carbohydrate load [40].

Holt et al. have noted that the glucose and insulin scores of most foods are highly correlated, but high-protein foods and bakery products that are rich in fat and refined carbohydrates “elicit insulin responses that were disproportionately higher than their glycemic responses” [40]. They also conclude that insulin indices may be useful for dietary management and avoidance of non-insulin-dependent diabetes mellitus and hyperlipidemia.

Glycemic Index (GI) considers each food relative to eating 100 % glucose, while the insulin index is relative to eating white bread (GI of ~70 to 75) (Table 6.5).

Table 6.5

Foods’ glycemic and insulin index list

	Glycemic index	Insulin index
Porridge	60 ± 12	40 ± 4
Muesli	43 ± 7	46 ± 5
Cornflakes	76 ± 11	75 ± 8
Average:	59 ± 3	57 ± 3
White bread (baseline)	71 ± 0	100 ± 0
White pasta	46 ± 10	40 ± 5
Brown pasta	68 ± 10	40 ± 5
Brown rice	104 ± 18	62 ± 11
French fries	71 ± 16	74 ± 12
White rice	110 ± 15	79 ± 12
Whole-meal bread	97 ± 17	96 ± 12
Potatoes	141 ± 35	121 ± 11
Eggs	42 ± 16	31 ± 6
Cheese	55 ± 18	45 ± 13
Beef	21 ± 8	51 ± 16
Lentils	62 ± 22	58 ± 12

6.1.5 Exercise

Exercise reduces insulin resistance by two mechanisms. It induces a reduction in visceral fat even if it results in moderate weight loss and BMI reduction [41]. Visceral fat is more metabolically active than subcutaneous fat and central adiposity is more closely related to IR [32].

Exercise, besides, increases muscle cell metabolism: it modulates the expression or the activity of proteins mediating insulin signaling in the skeletal muscles [41, 42].

It has been shown that exercise improves menstrual abnormalities and restores ovulation in obese patients with PCOS [43], and its benefit on reproductive function is greater than the benefit of low-calories diet only [44].

Exercise exerts its beneficial effects on body composition with a 45 % greater reduction in fat mass and a 60 % better preservation of fat-free mass [45].

In fact, it is important to clarify that improved abdominal obesity and insulin sensitivity may occur without a total change in body weight: body composition of patients who exercise regularly may change with increased lean body mass and decreased fat mass, but no overall change in weight [8].

At the moment, there are no guidelines for the type, intensity, frequency, and duration of exercise in patients with PCOS [45, 46].

6.1.5.1 PCOS Exercise Recommendations

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6.2 Insulin-Sensitizing Agents and Statins

Examining scientific literature, studies are very conflicting to each other, and a unanimous opinion on the effectiveness of insulin-sensitizing drugs has not yet been reached.

According to the ASRM Committee of 2008, insulin-sensitizing agents should be considered in patients with impaired glucose tolerance (IGT) and PCOS [47].

Particularly, in 2010, AE-PCOS Society consensus treatment emphasized that metformin should be used in women with PCOS who have already started lifestyle treatment (diet and exercise) and do not have improvement in IGT or in those who have normal weight but still having IGT [48].

When administered to insulin-resistant patients, these drugs act to increase target tissue responsiveness in order to reduce hyperinsulinemia [49].

In the past, limited studies on the use of diazoxide, acarbose, and somatostatin for PCOS women were conducted; then, thiazolidinediones aroused more interest, while, to date, metformin is the most worldwide studied insulin-sensitizing agent.

Moreover, statins have also been used to improve lipid profile in PCOS women.

6.2.1 Thiazolidinediones

Thiazolidinediones (TZDs) include pioglitazone, rosiglitazone, and troglitazone: during the past, they have been used in PCOS women to reduce insulin resistance.

TZDs are selective ligands of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) [50].

They exert their insulin-sensitizing actions by two mechanisms:

· Promoting fatty acid uptake and storage in adipose tissue

· Increasing the expression of adiponectin, an adipocytokine with an insulin sensitivity effect [51]

Obese women with PCOS who were administered troglitazone demonstrated benefit in insulin sensitivity, glucose tolerance, and hyperandrogenemia [52–56].

It was demonstrated that even pioglitazone, in doses of 30 mg/day for 3 months, significantly improved insulin sensitivity, hyperandrogenism, and ovulation rates [56].

TZDs were shown to be more effective than metformin in reducing levels of free testosterone and DHEAS after 3 months of treatment, but this benefit was not evident after 6 months of therapy [57].

Pioglitazone is able to produce a significant reduction in the incidence of impaired glucose tolerance and 40 % reversion of previous IGT to normal in PCOS patients treated with 45 mg daily for 6 months [58]. Significant improvements of insulin effectiveness in the liver and skeletal muscle, with substantial increase of circulating adiponectin levels, were also reported [59].

Moreover, some studies demonstrated a clear capacity of pioglitazone to reduce free fatty acid level in PCOS patients, by decreasing lipolysis and increasing lipogenesis [60]; conversely, other studies failed to show any improvement in lipid profile [61].

Additionally, some studies indicate a reduction of inflammatory markers in pioglitazone-treated PCOS women [62], while others do not [58].

A randomized study using treatment with pioglitazone showed that the latter increased ovulation frequency [63]. The regulation of ovulation could in turn restore normal feedback effects of luteal steroids, normalize serum LH levels, and improve ovarian steroidogenesis [64]. Additionally, pioglitazone was shown to ameliorate GnRH-stimulated LH secretion [56].

Administration of pioglitazone during ovarian stimulation period seems to improve ovarian response to controlled ovarian stimulation in PCOS patients, in terms of clinical pregnancy rate, as well as risks of ovarian hyperstimulation syndrome and multiple pregnancies [64].

Previously, TZDs have been accused of inducing weight gain and water retention, but recent studies have disconfirmed this supposition [65].

However, the primary concern with TZDs is the liver toxicity: a significant number of cases of hepatic necrosis were reported in patients using troglitazone, which was withdrawn from the market in 2000.

Pioglitazone safety in women under 18 is not yet established, so it is not recommended in this female PCOS subgroup.

However, in clinical practice, neither pioglitazone nor rosiglitazone is routinely used in PCOS women, especially with infertility issues, because TZDs are classified as pregnancy category C by the FDA, due to the fact that studies in animals have shown adverse fetal effects such as IUGR [64].

6.2.2 Metformin

Despite there is no universal consensus on metformin benefits in PCOS, in this chapter all the beneficial effects of metformin therapy in patients with PCOS are highlighted.

The positive effects of metformin have been demonstrated in nondiabetic women with PCOS, and they are associated with increased menstrual cyclicity, improved ovulation, and reduction in circulating androgen levels [66].

To date neither in Europe nor in the United States metformin has been approved for the treatment of insulin resistance associated with PCOS: its use should be restricted to those patients with IGT [67]; however, it is largely prescribed as an “off-label” drug.

For “off-label” use of any medication, it is extremely important to fulfill several criteria for safe use:

· The condition should have health consequences significant enough to warrant treatment.

· The treatment should have demonstrated safety and efficacy.

· The proposed treatment should be superior to the presently available alternatives [68].

6.2.2.1 Mechanism of Action

Metformin is a second-generation biguanide used as an oral antihyperglycemic agent, and it is approved by the US Food and Drug Administration (FDA) as treatment for type II diabetes mellitus.

It is considered an insulin-sensitizing agent because it lowers glucose levels without increasing insulin secretion, but improving insulin sensitivity.

Metformin causes [69, 70]:

· Increased peripheral insulin sensitivity, by activating glucose transporters (GLUTs) which allows passage of glucose into hepatic and muscle cells

· Inhibition of hepatic glucose production

· Reduction of circulating free fatty acid concentrations, which helps in reducing gluconeogenesis

Metformin activates the adenosine monophosphate (AMP)-activated protein kinase pathway (AMPK) [71, 72]: phosphorylation of threonine in AMPK is necessary for metformin action, resulting in decreased glucose production and increased fatty acid oxidation in hepatocytes, skeletal muscle cells [73], and mouse ovarian tissue [74].

Furthermore, metformin inhibits hepatic gluconeogenesis through an AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor small heterodimer partner (SHP) [75, 76].

Importantly, the actions of metformin are not associated with an increase in insulin secretion and, consequently, with hypoglycemia.

Metformin affects ovarian function in a dual mode:

· Alleviation of systemic insulin excess acting upon the ovary, particularly on steroidogenesis and follicular growth

· Direct ovarian effect

Furthermore, metformin acts at the hypothalamic level on AMPK pathway: the latter is essential in the modulation of LH secretion [77].

During the last two decades, some studies demonstrated that metformin inhibits androstenedione and testosterone production from theca cells through inhibition of the steroidogenic acute regulatory protein and 17α-hydroxylase expression [78].

At the ovarian level, hyperandrogenic intrafollicular pattern is improved by a decrease in IGF-1 availability that has an important role in controlling granulosa cell aromatase levels [79].

It has been shown that granulosa cells from women with PCOS have higher levels of FSH receptor (FSHR) expression compared with those from normal ovaries [80, 81].

Metformin reduces FSH-stimulated aromatase expression and activity in granulosa cells; it exerts this action by reducing FSHR mRNA and consequently the activity of FSH (as measured by aromatase expression and E₂), without altering cAMP levels. This involves blocking activation of CRE on promoter II of CYP19 via inhibition of pCREB and possible disruption of the formation of the CREB-CRTC2 co-activator complex. This is via an AMPK-independent mechanism [82].

6.2.2.2 Dosage and Side Effects

Metformin is available as 500, 850, and 1,000 mg tablets with a target dose of 1,500–2,550 mg/day.

Metformin has a dose-dependent absorption in humans [83], and its bioavailability is limited to 50–60 % because the amount available may result from pre-systemic clearance or binding to the intestinal wall [83].

Therapeutic regimens of metformin administration are not well standardized, and its dose should probably be adjusted according to the patient’s BMI and insulin resistance [84].

For example, it was demonstrated that nonobese women with PCOS respond better than obese women to metformin treatment at a dosage of 1,500 mg/day for 6 months. Nonobese women, in fact, showed a statistically significant decrease in serum androgen level and fasting insulin level and also an improvement in menstrual cyclicity [85, 86]. Moreover, it is possible that women who did not respond to metformin 1,5 g dose per day might show clinical changes if the dose is increased to 2 g [76].

Common side effects are gastrointestinal, such as diarrhea, nausea, vomiting, bloating, abdominal discomfort, flatulence, and unpleasant metallic taste in the mouth.

Lactic acidosis and hypoglycemia are very rare.

To reduce these side effects, it is recommended to start metformin with a low dose (e.g., 250–500 mg/day) and then gradually increase within a period of 4–6 weeks [76].

Metformin may cause vitamin B12 malabsorption, and so every patient should be monitored for signs and symptoms of vitamin B12 deficiency: numbness, paresthesia, macroglossia, behavioral changes, and pernicious anemia [66].

Metformin prescription should be avoided in women with renal insufficiency, congestive heart failure, sepsis, or hepatic dysfunction [66].

Therefore, testing of hepatic and renal function is necessary in advance of prescription, and thereafter yearly testing is indicated.

However, it has been demonstrated that metformin use for up to 6 months does not adversely affect renal or liver function in a large sample of PCOS women, even those with mildly abnormal baseline hepatic parameters [87, 88].

The length of metformin treatment in PCOS patients is not standardized, but data present in literature [89] showed that, after a long-term metformin treatment, drug suspension is related to a quick reversion of its beneficial effect on peripheral insulin sensitivity.

6.2.2.3 Metformin and Menstrual Disorders

The main complaint about menstrual disorders from PCOS patients is the absence or infrequency of menstrual bleeding.

Few studies noticed the regularization of menstrual cycle after 3–6 months of therapy with metformin alone in 60–70 % of PCOS insulin-resistant patients [90–93] with an important improvement of LH/FSH ratio [90].

The response to the treatment usually depends on the degree of insulin resistance.

The positive effect of metformin on menstrual cycle is commonly attributed to its effectiveness on ovulatory function. However, it is not uncommon to observe discordance between menstrual and ovulatory cycles.

The presence of ovulation should be confirmed through the measurement of luteal phase progesterone levels (usually, levels > 4 ng/mL indicate a previous ovulation) [76].

An Italian study revealed that only 79 % of PCOS women had ovulatory cycles after reaching normal menstrual cycle with metformin treatment [93].

This observation may indicate that the effectiveness of metformin on menstrual cyclicity is probably secondary to a direct effect on the endometrium and not only to an effect on the ovary [67].

Ovulation may be a result of a direct action of metformin on the ovary that leads to normal steroid production and steroid feedback effects that include a lowering of LH and androgen levels [67].

6.2.2.4 Metformin and Endometrium

Excessive insulin levels stimulate endometrial growth [94], and most anovulatory PCOS patients have endometrial vascularization and pattern and thickness abnormalities [95, 96]: PI (pulsatility index) and RI (resistance index) are higher than controls, probably due to the vasoconstrictive effect of androgens on vascular tissues [97].

Metformin may have a positive impact on the endometrium thanks to:

· Indirect effect: androgen decrease, which leads to the reduction of their vasoconstrictive effects on vascular tissue.

· Direct effect: insulin stimulates glucose oxidation activity in the late luteal phase in human endometrium; insulin receptors are present at the endometrial level, reaching their maximal expression in the secretory phase. GLUT-4 is an insulin-dependent transporter expressed in the endometrium and involved in endometrium metabolism; GLUT-4 is reduced in PCOS patients, suggesting that in these subjects both insulin resistance and hyperinsulinemia induce an inadequate GLUT-4 expression and so a decreased glucose supply. Thus, by improving hyperinsulinemia, metformin could be effective in restoring endometrial receptivity through a direct effect.

PCOS women who ovulated under metformin treatment showed a triple-line endometrial pattern in a percentage of cases similar to those observed in healthy controls [95], and a triple-line pattern is associated with a significantly higher pregnancy rate.

Another aim of metformin treatment is to reduce the long-term risks of unchallenged endometrial proliferation: hyperplasia and carcinoma.

Their main pathogenic mechanism assumed was hyper-estrogenic stimulation of endometrial growth, unopposed by progesterone. In fact, estrogens act by genetic and epigenetic mechanisms on cancer cells, and a close relationship between estrogens, growth factors, and oncogenes is important in the development of several human cancer [98].

The second hypothesis taken in consideration was the known mitogenic effect exerted by insulin [99].

6.2.2.5 Metformin and Hyperandrogenism

Metformin determines a great improvement on the hyperandrogenism symptoms of patients with PCOS, ameliorating hyperandrogenemia and reducing circulating insulin levels [92, 101–103]. Moreover, as insulin acts as an anabolic growth factor in hair [104], it is possible that the suppression of circulating insulin levels alone may be sufficient to improve the rate of terminal hair growth [76].

A 20–30 % reduction of total and free testosterone, increased SHBG levels, a 30 % decline of androstenedione levels, a modest decrease of FG hirsutism score, and an improvement of acanthosis nigricans were shown [92, 100].

Poor effects on the acne score of young PCOS women were recorded [105].

Several data suggest that metformin could act on hyperandrogenism by interfering both with direct and specific mechanisms on peripheral androgen-secreting organs and with free androgen fraction-regulating systems [67]: in fact, a reduced ovarian and adrenal secretion of androgens, a reduced pituitary secretion of LH, and an increased liver SHBG production seem to be the mechanisms that mediate metformin effect on hyperandrogenism [69].

On the other hand, other studies compared metformin effects to those obtained from oral contraceptives or antiandrogen drugs: the latter achieved a more effective results on hyperandrogenism than metformin alone [106–108].

According to our clinical experience, in overweight/insulin-resistant/hirsute PCOS women, metformin should be considered a first-line treatment, to be associated in combination with antiandrogen therapy.

Moreover, a case reported by an English study group demonstrated how important is the metformin administration even in underweight PCOS patients with menstrual disorder and hirsutism, underlying the essential role of insulin resistance in PCOS pathogenesis, sometimes independent of fat mass or distribution [109].

6.2.2.6 Metformin and Fertility

Metformin reduces insulin levels and alters its effects on ovarian androgen biosynthesis, theca cell proliferation, and endometrial growth; it inhibits ovarian gluconeogenesis, reducing ovarian androgen production [110–112]: all these actions lead to an improved ovulation induction in PCOS patients.

According to the ESHRE and ASRM guidelines issued in 2007, the use of metformin should be limited to patients with impaired glucose tolerance and should be interrupted before the administration of clomiphene citrate, thus restricting the use of metformin to a minority of PCOS patients [113].

However, more recent data suggest that these guidelines should be reconsidered.

Metformin alone has a significant benefit on inducing ovulation in PCOS women, but there is limited evidence that it improves pregnancy rate [101]. According to a multicenter study, metformin alone is not as effective as clomiphene citrate (CC) alone for the treatment of infertility: 55.3 vs. 75.1 % in cumulative ovulation and 7.2 % vs. 22.5 % of life birth [101].

On the contrary, an Italian study stated that the cumulative ovulation rate was similar in women treated with CC or metformin, whereas the pregnancy rate was significantly higher in women treated with metformin [114].

However, metformin is more effective than placebo alone, and it is associated with a significantly lower multiple pregnancy and ovarian hyperstimulation syndrome (OHSS) rate [76].

Because of the lack of evidence, metformin should not be used as first-line monotherapy, but only in those patients who:

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4.

In CC-resistant women, a combined therapy with CC + metformin (contemporarily or as pretreatment) is suggested: in a meta-analysis, this combination significantly improved ovulation and pregnancy rates, decreasing OHSS rate, when compared with CC alone [116].

The percentage of patients with PCOS and clomiphene resistance ranges in the different studies between 15 and 40 % [117, 118]. In these patients, metformin/clomiphene combination induces ovulation in 62.5–77.7 % of cases [116, 119–122].

This result is probably secondary to various mechanisms:

· Changes in intrafollicular steroidogenesis resulting from the effect of metformin on granulosa cells through an increase in insulin-like growth factor 1 [120]

· Inhibition of androgen synthesis by the direct action of metformin on the interna theca cells [78]

· Metformin-induced decrease of adrenal responsiveness to adrenocorticotropic hormone, resulting in reduced adrenal steroidogenesis [123]

· Reduction in serum LH and prolactin levels resulting from the effects of metformin on the hypothalamic–pituitary axis [124]

Thus, it is possible to state that metformin administration, decreasing insulin secretion, facilitates the induction of ovulation by using clomiphene citrate [125] in patients with PCOS.

The beneficial effects of metformin coadministration during gonadotropin ovulation induction and/or IVF cycles are unclear, and therapy with metformin should depend on the degree of IR.

It is well known that the response of PCOS women to gonadotropin stimulation differs significantly from that of normal ovaries: it is defined “explosive” and it is responsible for the higher risk of canceled cycles and/or for OHSS [126, 127].

In fact, it was shown that during ovarian stimulation, E₂ production and E₂-to-A ratio are higher in patients with PCOS who have elevated insulin levels than in normo-insulinemic women [126]. Increased insulin levels involve greater ovarian endocrine and morphologic responses to FSH-induced ovulation, which predispose to OHSS.

Therefore, it seems that the typical response of the polycystic ovary to exogenous gonadotropin therapy is related to increased plasma concentrations of insulin [128].

6.2.2.7 Metformin and Pregnancy Loss

Few observational studies have shown that metformin could play an important role in reducing the risk of pregnancy loss [129–131].

In particular, metformin exerts systemic actions by reducing body weight, insulin and PAI-1 levels [131–133], and plasmatic endothelin-1 (ET-1), androgen, and LH concentrations [135] and by increasing IGFBP-1 and glycodelin levels [136].

Moreover, metformin improved the uterine artery blood flow [95, 136] and several endometrial receptivity surrogate markers, as well as endometrial vascularization and pattern [95]. It was hypothesized that metformin might improve perifollicular and peri-corpus luteum vascularization too [95].

Furthermore, in the past, an experimental study [137] demonstrated that metformin also induced AMPK activation within the blastocyst, leading to improved insulin signaling and pregnancy outcomes. In fact, the preimplantation blastocyst stage embryo is an insulin-sensitive tissue, responsive to insulin or IGF-1 via the IGF-1 receptor/translocation of GLUT-4, with an increased glucose uptake [138]. High insulin or IGF-1 concentrations induced a downregulation of IGF-1 receptor [139] with consequent insulin-stimulated glucose uptake reduction, intraembryonic glucose level dropping, and apoptosis triggering [138].

On the contrary, other studies did not confirm these beneficial effects of metformin in preventing abortion [140, 141].

6.2.2.8 Metformin Administration During Pregnancy

The safety of metformin in pregnancy has not yet been established. It crosses the human placenta [142, 143], and it has been detected in umbilical cord blood at levels equal to or higher than the ones in maternal venous blood [144–146]: in fact, except for the first hours after metformin intake, the fetus is exposed to higher concentrations of metformin than the mother [144]. The knowledge on metformin metabolism in the fetus is scarce: it has been hypothesized that part of the metformin is excreted to the amniotic fluid [147] and reabsorbed to the fetal circulation by swallowing. Metformin is then eliminated from the fetus by passage through the placenta into the maternal circulation [144]. Fetal insulin concentrations are not affected by maternal metformin treatment.

A recent study demonstrated that intrauterine metformin exposure seems to result in elevated SHBG levels in newborns [148]. Metformin exposure throughout pregnancy exerts no major effects on maternal or neonatal androgens or estrogens at birth [148].

Metformin is classified as pregnancy category B [149]: a meta-analysis concluded that there was no evidence of an increased risk for major malformations [150].

Additionally, a study demonstrated that metformin did not adversely affect birth length, birth weight, growth, or motor–social development in the first 18 months of life [151].

However, current conservative practice would be to stop treatment once pregnancy has been established, but considering the adverse impact of insulin resistance on the pregnancy, continued metformin treatment after conception in women with PCOS may be beneficial [152].

The rationale of using metformin during pregnancy in PCOS women is the attempt to reduce the risk of developing gestational diabetes and other pregnancy complications associated with insulin resistance, such as preeclampsia.

Metformin seems to reduce the risk of gestational diabetes (GD) [153, 154] that complicates 5–40 % of pregnancy in women with PCOS.

Continued metformin treatment throughout pregnancy appeared to significantly reduce the rate of GDM requiring insulin therapy [155].

The mechanisms recognized in reducing GD incidence were the reduction of preconception weight, insulin, insulin resistance, insulin secretion, and testosterone levels and the persistence of these effects during pregnancy [156].

A less weight gain in women treated with metformin, compared with those treated with insulin, has been reported, and also the incidence of neonatal hypoglycemia was reduced [157, 158].

Furthermore, during the first trimester of pregnancy, metformin seems to influence the trophoblastic invasion of the maternal decidua, myometrium, and blood vessels, allowing a successful placentation with consequent pregnancy outcome improvement, such as prevention of pregnancy-induced hypertension (PIH) and preeclampsia [67].

Increased placental insulin resistance directly impairs nutrient supply to the fetus and leads to fetal growth restriction [159, 160].

Unfortunately, there are only a few studies in literature confirming these preliminary data.

Generally, it is important to note that the beneficial role of metformin in pregnancy-related parameters may be accomplished through a continuum of effects that starts from preconception and lasts throughout pregnancy [152]. In fact, preconception weight loss and IR reduction promoted by the combination of metformin and diet may reduce the likelihood of gestational diabetes in PCOS women [156].

Despite these favorable effects and reassuring clinical data, no definite guidelines recommending metformin use in pregnant women exist: further research is necessary [157, 161].

Finally, it is important to know that metformin is transferred into breast milk in amounts that appear to be clinically insignificant [162–165]. Thus, metformin use by breastfeeding mothers is considered safe. Nevertheless, each decision to breastfeed should be made after conducting a risk/benefit analysis for each mother and her infant [163].

6.2.2.9 Metformin and Metabolic Syndrome

As explained before, metformin increases insulin sensitivity [89, 100, 103, 166–169] and decreases weight, waist circumference, and BMI [100, 102, 167, 170], particularly if associated with diet and physical exercise.

Some authors state that, without metformin, weight loss (through caloric restriction and increased exercise) is difficult to achieve and maintain [171, 172], due to the weight-preserving and anabolic effects of high insulin [173] and androgens [91].

It was demonstrated that reduction of body weight, BMI, and visceral fat was greater than placebo, and the combination of metformin plus lifestyle intervention was more effective than placebo plus lifestyle intervention [174].

Metformin could act to improve body weight in obese PCOS patients both directly and indirectly:

· Direct effect: on the central nervous system, by modulating appetite in the hypothalamus [175]

· Indirect effect: via adipocytokine modification

Visfatin is the most recently identified adipocytokine, which seems to be preferentially produced by visceral adipose tissue and has insulin-mimetic action [176]. Circulating visfatin levels are higher in patients with PCOS than healthy controls, and it was demonstrated that metformin treatment significantly reduced visfatin levels after 3 months of therapy [177].

It has been suggested that weight loss may be a dose-related response with increased weight loss at higher dose [170]. In fact, comparing two different doses, a significant drop in BMI and waist circumference was seen in those patients using the higher dose [178].

Investigators have reported a greater weight, BMI, and WC reduction in obese patients receiving 2,550 mg/day and concluded that the long-term effect of metformin is better with greater dose [170, 179].

Additionally, metformin may slow the progression to type II diabetes mellitus [66].

This protective effect might be associated with the preservation of pancreatic beta-cell function and appeared to be mediated by a reduction in the secretory demands placed on beta cells by chronic insulin resistance [180].

A recent position statement from the AES (Androgen Excess Society) recommended that women with PCOS, regardless of weight, should be screened for IGT or type II diabetes mellitus by an oral glucose tolerance test at their initial presentation and every 2 years thereafter [181].

However, this statement noted that the use of metformin to treat or prevent the progression of IGT could be considered but should not be mandated at this point in time because well-designed RCTs demonstrating efficacy have yet to be conducted [67]. Moreover, it is important to underline that metformin does not maintain its benefits at a biochemical and clinical level after a 12-month treatment suspension [89].

It is widely known that insulin resistance and consequent metabolic syndrome increase the risk of cardiovascular disease: for this reason, it is very important to consider long-term health when selecting a medical treatment in overweight women with PCOS [182].

PCOS young patients usually do not manifest increased blood pressure values [183], but at menopause women with PCOS have a risk of developing hypertension 2.5-fold higher than age-matched controls [184]: metformin could prevent structural changes that precede hypertension [67]. In fact, it has been shown that metformin improve endothelial function, coronary microvascular function, and coronary flow rate [185].

As explained in previous chapters, dyslipidemia is a typical feature of metabolic syndrome: metformin improves hepatic fatty acid metabolism from lipogenesis toward oxidation.

Different beneficial effects are reported on dyslipidemia in PCOS women [130, 173, 186–192]:

· Decreased total and LDL cholesterol levels

· Decreased triglyceride levels

· Increased HDL cholesterol levels

To prevent vascular consequences, LDL particles should be normalized.

Despite metformin has been shown to improve metabolic alteration, it cannot be considered as first-line therapy [193], but it should be used as an adjunct to lifestyle modification.

Besides ameliorating the metabolic syndrome already present, metformin appears to be also effective in preventing the onset of the metabolic syndrome [194]; a study reported that PCOS women treated with a combination of metformin and controlled diet had significant and sustained improvements in all parameters of the metabolic syndrome over 4 years [195]. Conversely, another study showed that beneficial effects of metformin on the metabolic syndrome, without a specific lifestyle modification regimen, could be sustained over 3 years of routine clinic follow-up [194].

Furthermore, chronic inflammation is one of the PCOS features. Metformin alone reduces circulating levels of CRP (inflammation marker that is usually higher in PCOS women) [196]. It exerts a direct vascular anti-inflammatory effect by dose dependently inhibiting IL-1β-induced release of the pro-inflammatory cytokines IL-6 and IL-8 in endothelial cells, human vascular smooth muscle cells, and macrophages [67, 197].

Endothelial dysfunction, assessed by reduced flow-mediated dilatation, has shown promising results in cardiovascular risk stratification and prognosis [198, 199]. Metformin administration for 6 months in women with PCOS induced a significant increase in flow-mediated dilatation that was restored to normal values [200].

A recent study suggests that metformin decreases serum levels of asymmetric dimethylarginine (ADMA) levels, an endogenous inhibitor of NOS, by concomitant effects on insulin action and androgen levels [201].

Metformin seems to be effective even in decreasing AGE levels, which are oxidative mediators of endothelial dysfunction [134].

Plasminogen activator inhibitor-1 is a pro-thrombotic factor produced by the endothelium that inhibits fibrinolysis and regulates vascular smooth muscle proliferation [202]. Insulin upregulates PAI-1 gene transcription [203] and stimulates hepatic [204] and endothelial PAI-1 production [205]. It has been demonstrated that metformin reduces PAI-1 levels [131–133].

6.2.2.10 Metformin and Hypothyroidism

A recent study stated that in overweight PCOS patients with primary sub-hypothyroidism, treatment with metformin (1,500 mg/day) resulted in a significant fall in TSH and in some cases improvement of hypothyroidism [206].

This is an important finding because hypothyroidism occurs in more than 10 % of PCOS patients [207].

Several mechanisms have been hypothesized:

· A slight increase in the gastrointestinal absorption of levothyroxine (in patients already in treatment with L-thyroxine) [208].

· Influence of changes in body weight, associated with metformin therapy, on TSH levels [209].

· Increase of dopamine in the hypothalamus [210]. Previous studies, in fact, have suggested that there was a disruption of the neuroendocrine mechanisms in women with PCOS, mainly due to a deficiency in hypothalamic dopamine [211].

Further studies are needed to confirm these findings, but some authors suggest starting to treat obese PCOS patients with subclinical hypothyroidism with metformin and to reevaluate their thyroid function after 6 months [206].

6.2.2.11 Metformin Use in Lean PCOS Women

It was revealed that metformin decreases ovarian cytochrome P450c17α activity: this mechanism leads to a reduction of free testosterone serum levels even in lean PCOS women, with a consequent improvement of hyperandrogenism [212].

In fact, it has been demonstrated that women with PCOS who have normal weight or are thin responded to a reduction in insulin release with decreased ovarian androgen production and serum ovarian androgens.

As Nestler highlighted several years ago, metformin treatment of nonobese women leads to [213]:

· Decreased fasting and glucose-stimulated insulin levels

· Decreased basal and GnRH-stimulated LH release

· Decreased ovarian androgen production

· Decreased both serum total and free testosterone concentrations

· Increased serum SHBG concentrations

· Decreased androstenedione and DHEAS levels

Six months of metformin therapy clinically results in beneficial effects in lean PCOS women in terms of resumption of menses, without any remarkable effect on metabolic and cardiovascular risk factors [214].

Moreover, a very recent study suggests that treatment with metformin, for at least 12 weeks prior to, and during, IVF/ICSI, is worth considering as a management approach for nonobese women with PCOS [215].

6.2.2.12 Metformin Use in PCOS Adolescents

Metformin is indicated in patients older than 10 years, 2 years after their menarche.

Few studies demonstrated that metformin improved ovulatory function even in PCOS adolescents [93, 216], as well as hyperandrogenism [217].

As in adult population, metformin is effective in reducing hyperinsulinemia and lipid abnormalities.

A recent study reported that PCOS was the main indication for metformin prescription in UK general practice, even if it is off-label [218].

In the FDA approval process, several studies that demonstrated the safety of metformin use in the adolescent population were conducted.

Contraindications and side effects are the same described for adults.

Even if the literature on metformin in adolescents is limited and the number of studies inconsistent, it is possible that early intervention might prevent the complete spectrum of the syndrome in young overweight girls [219].

However, to date, in adolescent population, the first-step treatment is always the lifestyle modification.

In obese adolescents with PCOS, EP combination pills are the standard of care when lifestyle modification is not effective [220]. EP pills treat hyperandrogenism by increasing SHBG and so decreasing free testosterone; it reduces LH and FSH secretion and decreases ovarian stimulation and androgen production. Progesterone induces menstrual cyclicity and prevents endometrial hyperplasia [221]. However, EP pills do not treat IR or components of the metabolic syndrome [222] and are instead associated with glucose intolerance, decreased insulin sensitivity, abnormal lipid profiles, and CV disease [223, 224].

A recent study compared metformin monotherapy vs. estrogen–progesterone + metformin in the treatment of overweight and obese PCOS adolescents [220]: it was shown that the use of metformin alone was associated with greater decrease in total cholesterol and triglycerides and with a better improvement in weight loss.

These findings suggest that metformin monotherapy is more effective in reducing cardiovascular risk in overweight and obese adolescents with PCOS than the combination with EP pill [220].

6.2.2.13 Metformin + Pioglitazone in PCOS Treatment

Pioglitazone as add-on therapy in metformin-resistant PCOS women (e.g., in women who after 6 months’ 1,500–2,500 mg daily metformin treatment fail to improve their metabolic and hyperandrogenemia-related clinical signs) may exert beneficial metabolic (further reduction of IR and glucose levels, improved lipid metabolism, and lowering of carotid intima–media thickness) and antiandrogenic (improved menstrual regularity, significant drop in testosterone and DHEAS levels, increased SHBG, and improved hirsutism score) effects [64, 225, 226].

Moreover, the addition of pioglitazone was not associated with any adverse side effects, such as hepatotoxicity and hypoglycemia [226], providing another valid option for the management of NAFLD and NASH in PCOS women [64].

Pioglitazone safety in women under 18 is not yet established; thus, pioglitazone is not recommended in this female PCOS subgroup. Its safety in pregnancy and lactation is not entirely clear, but it is classified as category C drugs by the FDA due to the fact that studies in animals have shown adverse fetal effects such as IUGR [64].

However, long-term and large-sampled clinical trials are necessary before stating definitive conclusions.

6.2.3 Statins

Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the cholesterol biosynthesis [227]: inhibition of this enzyme decreases cholesterol synthesis with a compensatory increase in the expression of LDL receptors in the liver. In the general population, statins decrease total cholesterol and LDL cholesterol, and they have antiproliferative and antioxidant features on endothelial cells [228].

Statins reduce plasma triglycerides in a dose-dependent manner, and they also have a modest HDL-raising effect, which is not dose dependent [229, 230].

As dyslipidemia is a component of metabolic syndrome, atorvastatin and simvastatin have been used in PCOS women to investigate their effects on this common syndrome.

To date, there are limited data on the use of statins in PCOS, but short-term use of statins alone or in combination with metformin appears to improve lipid levels in PCOS. In a meta-analysis, statins were more effective than placebo in reducing total cholesterol, LDL, and triglycerides; lipid profile improvement occurred within the first 3 months of treatment, with no further significant change thereafter [231].

A combination of metformin with statins was more successful than metformin alone in lowering fasting glucose, fasting insulin, LDL cholesterol, and triglycerides [232].

Moreover, in the presence of simvastatin, metformin is much more effective in reducing testosterone, DHEAS, hirsutism, and LH and reversing the LH/FSH ratio in patients with PCOS [233].

The mechanisms of action of simvastatin on inhibition of T levels are likely related to the inhibition of the mevalonate pathway [233, 234]. Statins might also decrease the expression of several key enzymes involved in T production: cholesterol side chain cleavage (P450_SCC), 17α-hydroxylase/17,20-lyase (P450_c17), and 3β-hydroxysteroid dehydrogenase (3βHSD). Such effects of statins were noted in adrenocortical cells [235, 236]. The mechanisms of these actions might be due to the inhibitory effects of statins on isoprenylation [237], leading to decreased function of small guanosine triphosphatases, such as Ras: statin might abrogate Ras-induced steroidogenesis [236].

Additionally, statins induce inhibition of proliferation of theca interstitial cells and might reduce T output of the ovary by reducing the size of the theca interstitial compartment [233].

Thus, although simvastatin plus metformin could successfully reduce hyperandrogenism, insulin resistance, and lipid profile, its clinical significance is yet to be characterized [233].

However, statins are considered pregnancy category X drugs, and so it is always required to avoid contraception: this represents a very important restriction of use, and it is not a good option of treatment for all PCOS patients who want to get pregnant.

Finally, statins should be reserved only for women with PCOS who have increased LDL cholesterol [238].

6.3 Inositol and Other Supplements

In recent years, more attention has been paid to some supplements, which seem to have an important role in the therapy of PCOS, such as inositol and antioxidant molecules.

6.3.1 Inositol and Its Isomers

Several inositol isomers, and in particular myoinositol (MI) and D-chiro-inositol (DCI), were shown to have insulin-mimetic properties and to be efficient in the treatment of PCOS.

Inositol (cyclohexane-1,2,3,4,5,6-hexol) is a polyol existing under nine stereoisomeric forms depending on the spatial orientation of its six hydroxyl groups (Fig. 6.1).

Fig. 6.1

Inositol isomers

Myoinositol is naturally present in animal and plant cells, as free form, as inositol-containing phospholipid (phosphoinositides), or as phytic acid (IP₆) [239].

The greatest amounts of myoinositol in common foods are found in fresh fruits and vegetables and in peas, beans, grains, and nuts [240].

Originally, myoinositol was considered one of the B-complex vitamins, but now it is no more reputed an essential nutrient because it was shown that it is produced in sufficient amount in the human body from D-glucose [241].

It was shown that myoinositol is indispensable for the growth and survival of cells [242] and for the development and function of peripheral nerves [243]; it is essential to bone formation, osteogenesis, and bone mineral density [244], but its therapeutic implications are mainly related to its important role in glucose homeostasis.

A significant part of the ingested myoinositol is consumed in the form of phosphatidylinositol (PI) that may be hydrolyzed by a pancreatic phospholipase A in the intestinal lumen. Ninety-nine percent of the myoinositol ingested is absorbed from the human gastrointestinal tract, through an active transport system involving a Na⁺/K⁺-ATPase [239].

Cells mainly derive inositol from three sources:

· De novo biosynthesis from glucose-6-phosphate by 1D-myoinositol-phosphate synthase (MIPS) and inositol monophosphatase (IMPase)

· Dephosphorylation of inositol phosphates derived from breakdown of inositol-containing membrane phospholipids

· Uptake from the extracellular fluid via specialized myoinositol transporters [245]

In vivo, conversion of myoinositol to D-chiro-inositol can occur in tissue expressing the specific epimerase.

Myoinositol and D-chiro-inositol can also be bound components of glycosylphosphatidylinositol (GPI) anchors and of inositol phosphoglycan (IPG) that would constitute second messengers of insulin action in the GPI/IPG pathway [241].

The exact mechanisms of action of MI and DCI with insulin-mimetic activities are still unclear; a presumed mechanism of action implies inositol phosphoglycans (IPGs) containing MI or DCI as insulin mediators [241].

A few studies hypothesized that insulin, other growth factors, and classical hormones stimulated the hydrolysis of glycosylphosphatidylinositol (GPI) generating water-soluble inositol phosphoglycan (IPG) second messenger. The origin of IPG-A is thought to be myoinositol-containing GPI [246].

One of the most interesting models is the one elaborated by Larner and coworkers in 2010 [247]. According to this model, insulin binding to its receptor (IR) causes the autoactivation of the receptor, and the activated IR can transduce the signal through two parallel signaling pathways, which act together to mediate insulin action in a complementary and synergistic manner [241]:

1.

2.

The insulin-sensitizing effect of a MI and DCI supplementation is probably due to their intracellular enhanced availability for the production of membrane IPG precursors; numerous evidences support the hypothesis of a role of inositol glycan insulin second messengers in insulin-mimetic properties of some inositol isomers.

Moreover, it is known that part of MI supplementation effect on insulin sensitivity may come from its partial in vivo intracellular epimerization to DCI [241].

MI intracellular concentration is regulated through processes such as extracellular MI uptake, de novo biosynthesis, regeneration, efflux, and degradation. Alteration of one or several of these processes can lead to inositol intracellular abnormalities [241] in diabetes mellitus: inhibition of cellular MI uptake, altered MI biosynthesis, enhanced MI efflux due to sorbitol intracellular accumulation, and increased MI degradation are putative mechanisms of MI intracellular depletion [250].

Larner et al. noted a decreased urinary excretion of DCI and an increased urinary excretion of MI in humans and monkeys with type II diabetes (ten times higher compared to healthy subjects) [251].

The ratio of MI/DCI is regulated by an epimerase that converts MI into DCI [252], and Larner showed that each organ has a specific MI/DCI ratio [253].

Altered ratios of increased myoinositol to decreased D-chiro-inositol in urine have even been proposed as an index of insulin resistance in humans [254]: a deficit in MI to DCI epimerization activity, due to an epimerase-type enzyme, was supposed [249, 255].

Excessive urinary MI excretion could reduce MI plasma level and subsequently emphasize MI intracellular depletion, particularly in tissues heavily dependent on extracellular MI import [241]. Decreased production of DCI from MI reduces the availability of intracellular DCI for its incorporation into IPGs (particularly, DCI-IPG), probable downstream second messengers of insulin.

Furthermore, the decreased DCI content in insulin target tissues could reduce insulin signal transduction involving IPGs, in order to contribute to insulin resistance in those tissues. Depleted plasma levels of DCI observed in PCOS patients underline the correlation between impaired plasma DCI and insulin resistance [241].

Thus, insulin resistance is associated with:

1.

2.

3.

On the contrary, more recently (in 2006) Nestler proposed that, in a woman with PCOS, an initial genetic or environmental insult causing insulin resistance leads to a compensatory hyperinsulinemia. The latter induces a defect that increases renal clearance of DCI, and this leads to a reduction in circulating DCI and its availability to tissue. The consequence is an intracellular deficiency of DCI and of DCI-IPG, a mediator of insulin action.

Diminished release of DCI-IPG in response to stimulation by insulin results in a further decrease in insulin sensitivity [256] (Fig. 6.3).

Fig. 6.3

DCI alteration in insulin resistance, proposed by Nestler [256]

In 2010, Baillargeon et al. [257] showed that when plasma glucose is maintained at stable levels and plasma insulin is acutely raised and maintained at constant levels, the circulating DCI-IPG insulin mediator is released rapidly and briefly in normal women. Conversely, this coupling between insulin action and DCI-IPG release was entirely absent in obese women with PCOS: the release of bioactive DCI-IPG was significantly lower in obese PCOS women [257].

Possible explanations for these findings are a deficit in intracellular DCI or DCI-IPG and/or a defect in incorporation of the substrate DCI with membrane phosphoglycans to generate DCI-IPG mediator [257].

The possibility that a deficit in circulating DCI, or its precursor MI, is responsible for defective insulin-stimulated release of DCI-IPG mediator in PCOS is supported by the findings that oral supplementation with DCI [258–260] or MI [261, 262] to both lean and obese PCOS women improved their insulin resistance and clinical symptoms.

Moreover, defective DCI-IPG release in response to insulin could be due to a qualitative (rather than quantitative) defect in the insulin signaling mechanism that activates DCI-IPG mediator release from the membrane: there may be a primary defect in the union of the insulin receptor β-unit to the G protein or a defect in G-protein activation of phospholipase C [257].

This observation fits with Cheang et al. data [263]: they showed, in a number of hyperinsulinemic PCOS patients who did not respond to DCI treatment, the absence of changes in DCI-IPG release suggesting that a functional defect rather than a simple inositol nutritional deficiency might be present [263, 264].

6.3.1.1 Inositol as Treatment for PCOS

A supplementation with myoinositol or D-chiro-inositol was found to be safe and effective in improving metabolic and hormonal parameters in PCOS patients: the main mechanism of action is based on improving insulin sensitivity of target tissues, resulting in the reduction of insulinemia which has a positive effect on the reproductive axis and metabolism.

One of the first studies was conducted in 1999 by Nestler et al. [258], who found that the administration of D-chiro-inositol to women with polycystic ovary syndrome decreased the insulin response to orally administered glucose; simultaneously with the reduction in insulin secretion, women who received DCI had a significant improvement in ovulatory function and decreased serum androgen concentrations [258].

It was demonstrated in various studies that both DCI and MI are able to:

· Reduce LH levels, LH/FSH ratio, and testosterone levels [258, 265–268].

· Restore spontaneous ovulation and menstrual cycles [258, 260, 262, 265, 269].

· Improve cutaneous disorders of hyperandrogenism, reducing hirsutism and acne score [266].

· Decrease HOMA index [264, 265, 267].

· Reduce systolic arterial blood pressure [267, 268].

· Reduce leptin, LDL cholesterol levels, and triglycerides [269].

· Increase HDL cholesterol level [260, 269].

In view of all these findings, recently we conducted a research to evaluate the clinical, endocrine, and metabolic response of young women with PCOS, treated for 12 weeks with DCI.

From a clinical point of view, our study has highlighted a significant retrieval of menstrual cycle regularity (p < 0.001) in a rate higher than 60 % in patients treated and a significant improvement of acne score (p < 0.05) in patients with D-chiro-inositol treatment. Moreover, there was a significant decrease of triglyceride (p < 0.05) and basal insulin serum levels (p < 0.05) in patients treated with D-chiro-inositol [270].

Another clinical study of our group has shown that the administration of D-chiro-inositol in association with estro-progestins (0.03 mg of ethinyl estradiol and 3 mg of drospirenone) leads to a significant decrease of acne already from the second month of treatment, amplifying earlier the effects of oral contraceptives [271].

In literature no side effects after MI or DCI administration were reported when clinical dosage was used (max 1 g of DCI or 4 g of MI). Clinical trial data indicate that adverse events related to inositol treatment are gastrointestinal symptoms (nausea, flatus, loose stools, diarrhea) at a dose of 12 g/day or higher [272].

Moreover, MI or DCI supplementation was demonstrated to be effective in reducing the risk of gestational diabetes (GB) in PCOS women [273, 274], even if more studies are needed to confirm these preliminary data.

Finally, we suggest the use of MI (4 g die) or DCI (1 g die) as first-line treatment for those lean PCOS patients suffering from oligomenorrhea and mild hyperandrogenism. Combined therapy with diet, exercise, and metformin is reserved for insulin-resistant and overweight PCOS patients with oligomenorrhea and moderate hyperandrogenism.

6.3.1.2 Inositol and Oocyte Quality

Myoinositol function is also linked to the important role of IP3 in oocyte development and maturation [275, 276].

Oocyte cycle is usually arrested at metaphase of the second meiotic division. Calcium release mechanisms are shown to undergo modification during oogenesis, and maximal sensitivity of calcium release is acquired during the final stages of oocyte maturation: after fertilization, an increased level of intracellular Ca⁺⁺ occurs, and subsequent conclusion of meiosis [277, 278].

It was experimentally observed that immature oocytes (germinal vesicles or oocytes undergone in vitro process of maturation) contain a number of IP3 receptors less than those matured in vivo, leading to a reduction in Ca⁺⁺intracytoplasmic rise.

The disposal of Ca⁺⁺ from intracellular deposits is required for the oocyte’s activation that is manifested by the exocytosis of cortical granules, the perpetuation of the second meiotic division, the extrusion of the II polar body, the formation of two pronuclei, and the activation of protein synthesis from maternal RNA to prime the first mitosis.

Inositol depletion dramatically reduces transduction signal mechanisms mediated by IP3, altering the dynamics linked to the intracellular Ca⁺⁺ fluctuations.

Myoinositol supplementation may prevent this block and promote meiotic progression of the germinal vesicles; in fact, it was demonstrated that follicles containing high levels of MY, dosed in follicular fluid, present oocytes of good quality, and this may be related to a close correspondence between MI and inositol phosphates, necessary during oocyte maturation PIP2-mediated [279].

In human follicular fluid a greater concentration of myoinositol is a marker of good oocyte quality.

A recent clinical trial showed that only MI rather than DCI is able to improve oocyte quality [280]; the reason was explained by the “DCI paradox in the ovary” [281]: it is explained that “ovaries in PCOS patients likely present an enhanced MI to DCI epimerization that leads to a MI tissue depletion; this, in turn, could eventually be responsible for the poor oocyte quality characteristic of these patients” [282].

However, this hypothesis has yet to be confirmed: in fact, even DCI supplementation has shown a significant improvement in oocyte quality.

One of our recent studies showed that, in patients with PCOS, treatment with myoinositol and folic acid, compared to only acid folic treatment, reduces the number of germinal vesicles and degenerated oocytes at the time of oocytes’ pickup, without affecting the total number of oocytes retrieved. Moreover, an increased number of transferred embryos of good quality and a reduced amount of FSHR IU administered for the ovulation induction were shown [283].

These results were consistent with those found in other studies [284], suggesting the positive effect that myoinositol plays in the development of mature oocytes.

Furthermore, recent data demonstrate that by providing both MI and DCI in a physiological ratio (40:1), hormonal and metabolic imbalances are treated much more quickly compared to MI alone [252], especially in overweight PCOS patients who need to control insulin levels and increase ovarian MI content, reducing the risk of developing a metabolic disease [285, 286].

6.3.2 Antioxidants

Polycystic ovary syndrome is also associated with decreased antioxidant concentrations, and it is considered an oxidative state [287].

The decrease in mitochondrial O₂ consumption and GSH levels along with increased ROS production explains the mitochondrial dysfunction in PCOS patients [288]. The mononuclear cells of women with PCOS are increased in this inflammatory state [289], which occurs mostly in response to hyperglycemia and C-reactive protein (CRP) [290].

Physiological hyperglycemia generates increased levels of ROS from mononuclear cells, which activate the release of TNF-α and increase inflammatory transcription factor NF-kappa B. As a result, concentrations of TNF-α, a known mediator of insulin resistance, are further increased [290].

Oxidative stress and inflammation promotes hyperandrogenism, which augments the inflammatory load [289].

Oxidative stress promotes its effects causing damage to follicular proteins by the marking of free thiol groups [291].

Furthermore, reactive oxygen species (ROS) has been considered to play a critical role in the success of different IVF techniques. ROS are produced within the follicle, especially during the ovulatory process [292], and it is believed that oxidative stress may be a cause of poor oocyte quality [293]. In fact, high levels of oxidants, as H₂O₂, have been found in fragmented embryos [294].

MI and DCI are considered an effective therapy for PCOS women even for its antioxidant activity.

A recent study demonstrated that MI treatment positively affected the oxidative status of red blood cells (RBC), as shown by the partial restoration of GSH contents and the reduction of both band 3 Tyr-P levels and protein glutathionylation [295].

Moreover, there is evidence that melatonin plays an important role in the regulation of reproductive activity [296], and high levels of melatonin have been found in human preovulatory follicular fluid in concentrations that are almost threefold higher than serum levels [297–299]. It is known that melatonin and its metabolites are potent direct free radical scavengers [300–303] and indirect antioxidants, due to their ability to modulate gene transcription for antioxidant enzymes [304].

An Italian study demonstrated that, in patients undergoing IVF, treatment with melatonin plus myoinositol and folic acid reduced the number of germinal vesicles and degenerated oocytes and increased the number of top-quality embryos, compared to the therapy with only MI [305].

Other two important antioxidant molecules are SOD (superoxide dismutase) and ALA (α-lipoic acid).

Therapeutic strategy to reduce the oxidative stress includes diet rich in vegetables, weight reduction, physical exercise, smoking cessation, alcohol consumption reduction, and adequate number of sleeping hours.

6.3.3 Vitamin D

Vitamin D has pleiotropic effects on a large spectrum of intracellular regulatory processes, including insulin metabolism, or intrinsic apoptotic pathway, on both classical and nonclassical tissues, such as the ovary [306].

Moreover, as explained previously, calcium has an important role in follicle development, and both calcium and vitamin D deficiencies are considered as potential risk factors for insulin resistance and obesity [307–310].

Hypovitaminosis D was found in about 80 % of PCOS women [311, 312].

Supplementation of vitamin D (50,000 IU/week) and calcium (1 g/day) seems to support the positive effect of metformin therapy, with greater results in restoring normal menstrual regularity and improving hyperandrogenism symptoms, weight loss, and follicle maturation compared to metformin treatment alone [311].

Further studies are needed to confirm these data in order to use vitamin D + calcium supplementation as routine PCOS treatment protocol.

6.3.4 Glucomannan

Recently, glucomannan has been introduced as supplement for insulin resistance treatment.

Glucomannan is a high-molecular-weight polysaccharide obtained from tubers of Amorphophallus konjac: it consists of molecules of D-glucose and D-mannose, and it is soluble and absorbs water up to 200 times its weight.

Glucomannan exerts its activity by increasing the viscosity of food bolus during digestion: it creates a viscous gel that makes the bolus smooth and soft, and it forms a nondigestible coating around food particles.

This leads to a decreased time of food permanence inside the gastrointestinal tract: as main consequence, the action of digestive enzymes is partially avoided, resulting in reduced absorption of nutrients [313, 314].

Thus, glucomannan slows both lipid and carbohydrate absorption, reducing total and LDL cholesterol [315].

In diabetic patients, it is able to reduce postprandial glycemia and insulinemia [316].

Moreover, glucomannan increases the secretion of glucagon-like peptide 1 (GLP1), cholecystokinin (CCK), and peptide YY (PYY) [317], induces satiation and satiety [318], and preserves weight loss [319].

Minor adverse effects are normally GI related and include diarrhea, flatulence, and bloating.

Recently, an Italian study has shown that the association inositol–glucomannan may represent a good therapeutic strategy in the treatment of PCOS women with insulin resistance [320].

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