Physiological functions

In 1850, Johann Joseph Scherer isolated a hexahydroxycyclohexane from the muscle and he named this molecule “Inositol” .

In 1887, Maquenne established its cyclohexanol structure, purifying inositol from leaves . From the epimerization of the six –OH groups nine possible stereoisomers can be derived: cis-, epi, allo-, myo-, neo-, scyllo-, L-chiro-, D-chiro-, and muco-inositol . Among them, myo-inositol (MI) is quite ubiquitous in nature and is the most abundant form found .

In the human body, MI synthesis takes place in the kidneys, brain, liver, testes and mammary gland. In particular, kidneys produce daily approximately 4 g MI .

MI derives from the isomerization of glucose-6-phosphate (G6P) to inositol-3-phosphate (Ins3P) by D-3-myo-inositol-phosphate synthase enzyme (inositol synthase, Ino1, or MIPS1) . Then, through inositol monophosphatase-1 (IMPA-1 or IMPase), Ins3P is dephosphorylated to free MI .

Free inositol could also be obtained by the dephosphorylation of inositol-1,4,5-trisphosphate (InsP3) and inositol-1,4-bisphosphate (InsP2).

A specific Nicotinamide Adenine Dinucleotide (NAD)-NADH-dependent epimerase, under insulin stimulation, converts MI into D-chiro-inositol (DCI) .

Also, a normal diet provides inositols, mainly present in fruits, beans, grains, nuts cereals and legumes, as MI for the most part, phosphatidylinositol (PI) and inositol hexakisphosphate .

In order to exert its activity, insulin needs the presence of both MI and DCI. The two stereoisomers, as inositol-phosphoglycans (MI-IPG and DCI-IPG), are second messengers in the insulin signaling, mediating different effects . The activation of glucose transporters and glucose utilization are controlled by MI, whereas DCI regulates glycogen synthesis . Both molecules are therefore able to exert an insulin-sensitizing effect reducing the circulating insulin levels.

Furthermore, specifically, MI participates in the follicle-stimulating hormone (FSH) signaling, acting as second messenger. Through its receptor (FSHR), FSH regulates the proliferation and maturation of granulosa cells. MI-IPG also seems to regulate the cytoskeleton and the FSH-induced production of anti-Müllerian hormone (AMH), which modulates the sensitivity of follicles to FSH and, hence, their maturation .

On the other hand, DCI, in the phosphoglycan form, besides acting as a second messenger of insulin signal indirectly influences steroidogenesis. Nevertheless, DCI also exhibits an independent activity on androgen biosynthesis, as found by Nestler and co-workers . They demonstrated that DCI can reduce the activity of the enzyme aromatase (CYP19A1), that catalyzes the conversion of androgens to estrogens. These results are supported by the study of Sacchi and colleagues; they found that DCI affects the gene expression of aromatase, downregulating it in a dose-response manner .

Thus, DCI activity on steroidogenesis, may be twofold: on the one hand, an indirect effect as a result of insulin signal boosting; on the other hand, an independent direct effect on steroid biosynthesis through downregulation of the aromatase expression and enhancement of testosterone production .

Inositol’s effects in PCOS

Over the years, the critical role of insulin resistance and/or compensatory hyperinsulinemia in the pathogenesis of PCOS have been consistently affirmed throughout the numerous in vitro and in vivo studies.

Approximately 35% of lean women and 80% of obese women with PCOS present the condition of insulin impairment .

Cheang and colleagues confirmed that an impairment of the inositol-phosphoglycan insulin second messenger pathway might cause a derangement in the insulin signaling in PCOS . Therefore, the rationale for the use of these two molecules as valuable therapeutic approach of PCOS is designated primarily for their “insulin mimetic” action.

Treatment with inositols proved to be side effect free, at the therapeutic dose, and effective in improving several clinical features of PCOS. Of note, FDA included MI in the list of specific substances defined “generally recognized as safe” (GRAS) .

“Vitamin D”: Beyond the bone

Vitamin D is the old name used for a secosteroid hormone well known for maintaining calcium homeostasis and promoting bone mineralization . More appropriately, we should use the name of vitamin D only when this molecule is administered exogenously in cases of hormone D deficiency .

To become active, it requires two hydroxylation steps, yielding first calcidiol (or calcifediol), 25(OH)D, and eventually calcitriol, 1,25(OH)2D. The former is the most long-lived, abundant form of hormone D in the bloodstream, whereas the latter is the short-lived, most active form .

Beyond the established relationship between hormone D deficiency and musculoskeletal diseases, a growing body of literature suggests mechanistic implications of hormone D deficiency for insulin resistance, inflammation, dyslipidemia and decreased fertility, namely clinical and metabolic phenomena commonly encountered in PCOS .

Additionally, positive associations are reported between hormone D and some well-known comorbidities of PCOS, including type 2 diabetes, metabolic syndrome, and cardiovascular diseases .

In support of this stated above, hormone D modulates glucose-insulin homeostasis through the action on its own specific receptor VDR, located on the pancreatic β-cells and in the skeletal muscles. It directly activates the transcription of the human insulin receptor gene, activates peroxisome proliferator activator receptor-δ, stimulates the expression of insulin receptor, and enhances insulin-mediated glucose transport in vitro .

Low levels of hormone D appear correlated with hirsutism, hyperandrogenism and obesity. A recent metaanalysis found that prevalence of hormone D deficiency was 35% higher in obese and 25% higher in overweight compared with lean subjects. In addition, hormone D plasma levels are inversely correlated to all the parameters of obesity, including BMI, fat mass and waist circumference .

The fact that adipose tissue is the main storage site for hormone D and/or its metabolites in the body has prompted the hypothesis that this hormone and/or its metabolites gets sequestered in the excess fat mass in obese persons . However, the physiological mechanisms underlying this hypothesis have not been brought forward.

Nevertheless, as reported in a recent study from Drincic et al , it could just be that in individuals with a higher body mass, hormone D is simply diluted in a higher volume, so these subjects would require greater vitamin input than lean individuals to achieve a sufficient hormone D status. Decreased plasma levels could also result from a modification in hormone D metabolism occurring during obesity development. Indeed, modifications in the expression of genes encoding key enzymes of hormone D metabolism have been reported in the adipose tissue of obese people .

Finally, hormone D plays a physiological role in reproduction by modulating follicular development through the influence on the signal of the anti Müllerian hormone (AMH), on FSH sensitivity and on the production of progesterone in the ovarian granulosa cells .

Hormone D and progesterone: An interesting overlapping

Several studies reported a functional overlapping between hormone D and progesterone, likely due to the similarity in their chemical structure .

Progesterone prepares the environment conducive to pregnancy success and it is mandatory, during the secretory phase of menstrual cycle, to induce the endometrium receptivity to the embryo implantation .

The role of hormone D in fertility and reproductive capacity has been investigated in animal models for many years, demonstrating that hormone D-deficient female rats had reduced fertility rates . VDR knockout female mice were unable to reproduce due to defects in uterine development. Furthermore, the role of hormone D in uterine physiology seems to be essential for the normal differentiation of decidual cells .

Also, in human, the relevance of hormone D activity for pregnancy, from the luteal phase, has been confirmed by several studies. Recently, Bezerra Espinola et al. showed that the supplementation of vitamin D3 in the evening from the early beginning of the luteal phase positively correlated with the implantation rate in IVF .

As a matter of fact, especially in the first months of gestation, adequate levels of vitamin D are even more essential, affecting not only the well-being of the mother but also that one of the fetus. Insufficient levels of hormone D have been related to the pathogenesis and the abnormal development of luteal phase, leading to major risks of miscarriage and recurrent pregnancy losses for the pregnant women.

Even though hormone D and progesterone bind their own receptors and exert their specific biological functions, the two steroid hormones are able to cooperate with each other in immunoregulation .

Indeed, while implantation and labor take place in an inflammatory environment, the rest of gestation requires noninflammatory conditions, otherwise premature pregnancy termination can occur .

The main progesterone-influenced components to immune adaptation during pregnancy include an altered cytokine balance, reduced T cell responses and natural killer cells (NK) activity, as well as increased activity of regulatory T cells .

Hormone D, acting also as an immune-regulatory steroid hormone, suppresses Th0 maturation into Th1 and Th17 cells, favoring T-cell differentiation to Treg cells .

The efficacy of Treg cells in immune suppression is higher when they are produced under the combined effect of hormone D and progesterone than when there is only one of these effector molecules.

As previously stated, the biological actions of hormone D are mainly mediated by VDR, belonging to the nuclear receptor superfamily, and expressed in different organs and tissues including skeleton, immune system, parathyroid glands and reproductive tissues .

According to the reproductive tissues, VDR is present in the ovaries, uterus and placenta and it is expressed in the endometrial stromal cells throughout the entire menstrual cycle and in early pregnant decidual cells .

On the endometrium, the binding of hormone D to VDR upregulates the expression of calbindin and osteopontin , essential proteins for embryo implantation, placenta development and for successful pregnancy.

Considering this overlap between hormone D and progesterone in immunoregulation, it has been evaluated if these two hormones could act synergistically, and recently, Thangamani et al. have reported a more effective T cells regulation by hormone D when progesterone is present at the same time .

During pregnancy, progesterone induces in lymphocytes the expression of a protein called progesterone-induced blocking factor (PIBF), which mediates some of its immunological effects on cytokine balance and on NK activity .

Indeed, the presence of PIBF alters the Th1/Th2 balance favoring Th2 with 8–10 times increased production of antiinflammatory cytokines (IL-10, IL-4, and IL-5) while Faust et al. demonstrated that PIBF inhibits the cytotoxicity of peripheral NK cells via a block of degranulation .

PIBF is present in pregnancy serum as well as in the urine of pregnant women, and its concentration is predictive of the outcome of pregnancy. A study compared PIBF concentration in urine samples of 500 pregnant women and 80 nonpregnant individuals.

During a healthy pregnancy PIBF concentration in urine samples continuously increases until the 37th gestational week, starting to decrease thereafter, to disappear when labor starts. In women with threatened miscarriage or threatened preterm delivery, urinary PIBF levels remain significantly lower than those of healthy pregnant women of corresponding gestational ages .

Unpublished preliminary data indicated that, similarly to progesterone, hormone D can induce PIBF production in activated human peripheral lymphocytes. PIBF inducing capacity of hormone D was significantly higher in lymphocytes obtained during the luteal phase than in those of the follicular phase, suggesting that hormone D acts more efficiently in progesterone-primed lymphocytes. In line with this, peripheral lymphocytes treated with increasing concentrations of vitamin D plus a standard concentration of progesterone produced significantly more PIBF than those treated with similar concentrations of vitamin D alone.

These evidences indicate that progesterone and hormone D act in a combined manner, mutually potentiating their antiinflammatory activities to support early pregnancy.

Clinical evidence of vitamin D effectiveness in PCOS

In PCOS women hormone D deficiency seems to be three times less likely to carry on a pregnancy, and that supplementation may significantly revert this outcome.

In a posthoc analysis, Pal and co-workers examined the relation between hormone D status and pregnancy outcomes in PCOS patients. The authors found that ovulation correlated with hormone D levels. More specifically, they reported that the likelihood for live birth was reduced by 44% in women with low levels (< 30 ng/mL) of 25 hydroxyvitamin-D (25OH-D), whereas there was an improvement in the odds for live birth at the following thresholds: > 38, > 40, and > 45 ng/mL.

Serum 25OH-D was significantly higher in women achieving a live birth compared to those who did not, underlining a correlation between hormone D levels and fertility and suggesting possible beneficial effects of vitamin D supplementation in PCOS women with low serum levels of 25OH-D .

On these bases, researchers focused on vitamin D supplementation for the treatment of patients with PCOS. In their study about vitamin D replacement, Selimoglu and colleagues administered vitamin D3 for three weeks in 11 subjects with PCOS, evidencing some beneficial effects on IR, while no changes in androgen levels were observed .

The metaanalysis by Łagowska evaluated the effect of vitamin D supplementation on insulin resistance in PCOS patients. The authors concluded that the supplementation in PCOS patients with low doses of vitamin D (from 200 to 4000 IU/day) may improve insulin sensitivity in terms of the fasting glucose concentration and HOMA-IR.

Fertility and reproductive parameters were evaluated in another randomized double-blind, placebo-controlled trial . Ninety insulin resistant women with PCOS were assigned to three groups to take either 4000 IU of vitamin D, 1000 IU of vitamin D or placebo daily for 12 weeks. Compared with placebo, significant increases in SHBG and in modified Ferriman-Gallwey scores occur already at the lower dose of vitamin D.

In a double-blind, randomized placebo-controlled trial, Trummer and colleagues randomized 180 PCOS women to receive either vitamin D (20000 IU/week) or placebo for 24 weeks . Supplementation with vitamin D led to a decrease in plasma glucose 1 h after the oral glucose tolerance test (OGTT), compared to the placebo.

Fatemi et al. investigated the role of supplementation with vitamin E and vitamin D3 on ICSI outcomes in women with a PCOS diagnosis . The RCT included 105 infertile PCOS women randomized to receive either a combination of vitamin E and vitamin D3 (vitamin E, 400 mg/day and vitamin D3, 50,000 IU/two weeks, n = 52) or placebo ( n = 53) for 8 weeks after ICSI procedure. Implantation rate, as well as biochemical and clinical pregnancy rates were evaluated as primary outcomes. At the end of the study, all primary outcomes significantly improved in the supplementation group with respect to placebo (biochemical pregnancy rate: 69% vs 25.8%; clinical pregnancy rate: 62.1% vs 22.6%; implantation rate: 35.05% vs only 8.6%).

The prospective study by Zhao et al. evaluated the IVF/ICSI outcome in 305 infertile PCOS patients, according to their hormone D status .

The authors scheduled the patients into four groups: deficiency group without treatment, normal group without treatment, normal group after treatment and deficiency group after treatment. The observed clinical pregnancy rates, respectively of 19.3%, 65.2%, 66.7%, and 23.5%, indicate that patients with hormone D deficiency are about 3 times less likely to carry on a pregnancy following the IVF/ICSI procedure, and that supplementation significantly improved this outcome.

In contrast with the finding of Fatemi et al , the authors also observed particularly marked differences in the number of high-quality embryos and available embryos for transfer. They concluded that vitamin D supplementation restores serum levels of vitamin D in infertile women with PCOS and IR, leading to improved embryo quality and to significantly higher clinical pregnancy rates.

Dastorani et al. suggested that improved metabolic profile of lipids and insulin may account for the beneficial impact that vitamin D supplementation has on IVF/ICSI outcomes in PCOS women . In this randomized, double-blinded, placebo-controlled trial, 40 infertile PCOS women were assigned to 2 groups to receive either vitamin D (50,000 IU) or placebo ( n = 20 each group) every other week, for 8 weeks. The authors observed that vitamin D supplementation resulted in a significant reduction in serum AMH, insulin levels and homeostatic model of assessment for insulin resistance, while significantly increasing the quantitative insulin sensitivity.

All these results highlighted the positive effects of vitamin D supplementation on different pathologic features of PCOS. Nevertheless, more studies are needed to ascertain the benefits of vitamin D supplementation in PCOS management, also in association with other molecules.

Physiological functions

Recently, also alpha-lipoic acid (ALA) has been considered a possible therapeutic approach to PCOS and IR .

ALA and its reduced form, dihydro-lipoic acid (DHLA), are powerful antioxidant molecules with reactive oxygen species (ROS) scavenger function and able to regenerate other antioxidant molecules . Moreover, ALA is an inhibitor of the inflammatory pattern mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) , and it also has an immunomodulatory function .

In the metabolic field, ALA improves insulin sensitivity promoting the expression of 5′-adenosine monophosphate-activated protein kinase (AMPK), a cellular energy sensor that induces the translocation of glucose transporter 4 (GLUT4) to the plasma membrane with an insulin-independent mechanism . A reduced ALA synthesis, probably due to the downregulation of the lipoic acid synthase that occurs during diabetes mellitus (DM) and IR, is supposed to affect the normal glucose uptake and utilization in skeletal muscle cells .

Clinical evidence of ALA in PCOS

Some scientific evidence suggests that ALA may improve reproductive function and metabolic parameters in women affected by PCOS.

Masharani and colleagues administrated a preparation of controlled release ALA 600 mg twice a day for 16 weeks in a group of 6 lean women affected by PCOS. Despite the absence of severe insulin resistance in this group of patients, therapy with controlled-release ALA led to a lowering of triglyceride levels, improvement in insulin sensitivity and menstrual frequency .

Few years later, Genazzani and colleagues observed that a combination of 400 mg of ALA with inositol (1 g MI) reduced IR and glucose-load induced hyperinsulinemia in a group of 36 PCOS patients, also improving gonadotropin secretion . ALA plus MI, in addition to treatment with metformin, also showed a better response in terms of hyperandrogenism, BMI and HOMA index than metformin alone in women with PCOS .

Also, treatment with a combination of 1 g DCI and 600 mg ALA daily for 180 days versus no treatment in a group of 46 women of reproductive age with PCOS led to encouraging results in terms of clinical and metabolic features. In fact, in the treated group HOMA-IR, insulin levels, lipid profile and frequency of menstrual cycles were significantly improved .

These results are in line with those ones from Fruzzetti and colleagues on 41 women with PCOS and IR . They observed that the association of DCI and ALA was effective in improving BMI, insulin sensitivity and menstrual disorders, though some limitations such as the absence of a placebo group and the use of the OGTT and HOMA calculation instead of the hyperinsulinemic glycemic clamp to test insulin sensitivity.

Overall, though presented data suggest that the association of inositols and ALA may be a viable way to manage PCOS, assuring a normalization of menstrual cycles and an improvement of insulin secretion along with high tolerability, however other studies need to be carried out to clarify the rationale of the association between ALA and inositols. In fact, while the inclusion of the protein alpha-lactalbumin to MI has its therapeutic rationale, as shown before, combining ALA with MI could be questionable since we do not have studies on the pharmacokinetics of ALA nor do we know if this association with MI could have too long-term drastic effects on glucose metabolism .