Inositols and other supplements in the management of PCOS





Introduction


Polycystic ovary syndrome (PCOS) is associated with neuroendocrine dysfunctions, insulin resistance (IR), impaired ovarian steroidogenesis and hyperandrogenism .


Although the etiology of PCOS is still poorly known, androgens and insulin are thought to be two key factors in its pathogenesis. Thus, PCOS treatment needs to cure both hyperandrogenism as well as hyperinsulinemia.


Given the central role of insulin resistance in the onset of PCOS, insulin-sensitizing agents, such as metformin and pioglitazone have been proposed as first line approaches . However, pioglitazone though it positively modulates ovarian androgen synthesis via upregulation of progesterone biosynthesis, inhibition of testosterone, and production of E2, may be associated with relevant adverse effects , and metformin has only convincingly demonstrated the advantage of lowering IR, while women affected by PCOS present very often other metabolic and reproductive disorders .


Therefore, such a heterogeneity of clinical manifestations of PCOS suggests that the therapeutic strategy should consider the overall features of the patient and include pharmacological and/or nonpharmacological treatments.


This chapter will be focused on different supplements that may help to manage PCOS and its complications, starting with inositols.


Inositols


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) .


Clinical evidence of MI effectiveness


As evidenced by Papaleo and co-workers, a 6-month treatment with 2 g MI twice a day, effectively restored spontaneous ovarian activity, and subsequent fertility in PCOS patients . In 72% of these patients, MI reactivated the normal ovulatory activity and achieved a pregnancy rate of 40% during the 6-month observation period.


Other researchers replicated these results investigating the metabolic and hormonal effects of MI in PCOS women. Patients underwent hormonal evaluations and OGTT before and after treatment; ultrasound examinations and Ferriman-Gallwey score were also performed. After MI administration, plasma LH, prolactin, testosterone, insulin levels and LH/FSH ratio were significantly reduced and insulin sensitivity, expressed as glucose-to-insulin ratio and HOMA index, resulted significantly improved. Menstrual cycle was restored in all subjects with amenorrhea or oligomenorrhea.


Thus, hormonal profile significantly improved, and the restoration of ovulation as well as regular menses in both obese and lean women was observed .


Up to date, several studies have been conducted on the effect of inositol on the biochemical and clinical parameters related to hyperandrogenism and metabolism in PCOS women .


Overall, these studies, even with many differences in trial design, sample size, treatment duration and dosage, share a common result: the beneficial effect of MI alone or combined with DCI in improving the metabolic profile of women with PCOS, and a concomitant reduction of hyperandrogenism .


Clinical evidence of DCI effectiveness


PCOS women have reduced serum level and increased urinary loss of DCI . This could be due to multiple causes, including a defective conversion of MI to DCI and/or to impairment of tubular transport by high glucose.


DCI administration re-stablishes an adequate tissue content of DCI derivatives, increasing insulin sensitivity and improving ovulatory frequency and serum androgens and/or levels of lipid biomarkers in women affected by PCOS .


The first study with DCI versus placebo in PCOS was published in 1999 by Nestler and colleagues. They orally supplemented 1200 mg of DCI once a day to 44 obese PCOS women; after 6–8 weeks an improved insulin sensitivity and a decreased circulating free testosterone level were observed. DCI administration also resulted in ovulation for 86% women, whereas only 27% ovulated in the placebo group .


In 2002, the same group performed a follow-up study in lean women with PCOS . Again, and in agreement with the earlier study , the administration of DCI was associated with improved insulin sensitivity, a reduction in circulating free testosterone, and increased frequency of ovulation.


Subsequently, Insmed Pharmaceuticals embarked on a large multicenter placebo-controlled trial supplementing PCOS woman with 2400 mg DCI, a dosage twice as high as ever previously used. However, the higher dose of DCI failed to reproduce the outcomes of the two previous studies in terms of improving ovulatory frequency; thus, the results were never published. Consequently, the company gave up proceeding with the use of DCI in clinical trials on PCOS.


The higher dose of DCI administered was identified as the main cause of the lack of efficacy in the latter trial.


Further evidence that DCI administration to PCOS women can improve insulin sensitivity and reduce serum free testosterone levels, leading to normal cycle and ovulation, is available from a retrospective study performed in PCOS patients with irregular cycles. In this case, 1–1.5 g DCI administered daily for a maximum of 15 months improved insulin levels along with an increase in the percentage of women reporting regular menstrual cycles, directly proportional to the duration of the treatment .


In another study, 1 g DCI plus 400 μg folic acid daily for 6 months significantly improved IR as measured by HOMA index and glycaemia/insulin resistance index (IRI) ratio. In the same study, an improvement of systolic blood pressure, Ferriman-Gallwey score, LH, LH/FSH ratio, total testosterone, free testosterone, 1-4-androstenedione, prolactin, and sex hormone binding globulin was observed .


Despite this evidence showing positive effects of DCI in the treatment of PCOS, Legro in 2016 reported that large, multicentred phase 2 clinical trials were suspended because of lack of efficacy .


Interestingly, in a comparative study of the effects of administration of MI versus DCI on oocyte quality in PCOS patients, Unfer and coworkers observed that in the MI group the number of mature oocytes was significantly higher, with a parallel diminution in the number of immature oocytes, in respect to the DCI group .


This phenomenon is likely to be related to the tissue-specific nature of insulin resistance in women with PCOS. Indeed, meanwhile, fat, muscle and liver are insulin resistant in women with PCOS, ovaries never become resistant. Unfer and coworkers refer to this phenomenon as “DCI paradox” in the ovary .


In fact, since in the ovary, under insulin stimulation, the epimerase converts MI to DCI, Unfer et al. proposed that in PCOS women hyperinsulinemia overstimulates ovarian epimerase activity, resulting in a harmful increased production of DCI and a concomitant depletion of MI. The authors postulated that the resulting deficiency of MI could be responsible for the poor oocyte quality and the impairment of the FSH signaling. Clearly, DCI supplementation would be ineffective, if not harmful, in such women as they already have high levels of this molecule in the ovary.


Preclinical and clinical evidence of effectiveness of MI/DCI combination


In physiological conditions the plasmatic ratio MI/DCI is 40:1. This ratio could represent the best choice to restore ovarian cells function increasing MI content and concomitantly taking advantage of DCI function. Indeed, the 40:1 ratio, rather than MI or DCI separately, shows better result for counteracting both hyperinsulinemia and hyperandrogenism in PCOS.


The first clinical evidence of effectiveness of the 40:1 MI:DCI ratio has been provided in 2012 by Nordio and colleagues . They enrolled 50 overweight women with PCOS and divided them in 2 groups to receive MI plus DCI in the 40:1 ratio or MI alone for 6 months. At the end of the treatment, they observed that both MI and MI plus DCI treated groups showed an improvement of the metabolic parameters. Importantly, as expected, the combined supplementation with MI and DCI resulted to be more effective, compared to MI-treated group, after three months of treatment.


Just 1 year later also Minozzi and co-workers observed that the combined therapy with MI and DCI in the 40:1 ratio reduced the cardiovascular risk by improving the lipid profile in 20 obese PCOS patients, confirming the effectiveness of the association between the 2 isomers.


These results were also corroborated by the study from Benelli and colleagues on 46 patients affected by PCOS randomly assigned to two groups. In group A, 21 women received MI plus DCI combined treatment at the ratio of 40:1 in soft gel capsule containing 550 mg of MI, 13.8 mg of DCI, and 200 μg of folic acid twice a day. Group B, with 25 women, received the same amount of folic acid (200 μg) as placebo twice a day . The treatments were performed for 6 months.


The authors showed a reduction of LH, free testosterone, fasting insulin, and HOMA index only in the group treated with the combined therapy of MI and DCI in the 40:1 ratio; moreover, in the same patients they observed a statistically significant increase of 17-β-estradiol levels, concluding that the combined therapy of MI plus DCI was effective in improving endocrine and metabolic parameters. Though all these clinical evidence about how the 40:1 MI to DCI ratio was more effective in improving PCOS features, the first preclinical demonstration of the effectiveness of this ratio compared to other ratios has been provided only in 2019 by Bevilacqua and co-workers . They used an animal model of PCOS: female mice were exposed to continuous light for 10 weeks, developing an androgenic-like phenotype of the ovaries as in PCOS women. The authors provided the first experimental evidence that the efficacy exerted by various MI/DCI molar ratio (5:1; 20:1; 40:1; 80:1) changes, supporting the metabolic link between the two stereoisomers, specifically for PCOS.


The daily treatment of mice with 420 mg/kg MI/DCI in a 40:1 molar ratio allowed to obtain a rapid and almost full recovery from PCOS signs and symptoms.


Since the hypertrophy of the theca cell layer is a hallmark of PCOS, strongly associated to an increased production of androgens , it is noteworthy that the ovaries from treated mice recovered normal histological features, with the reduced ratio between theca and granulosa cell layer thickness. This means that the androgenic phenotype was efficaciously reversed. The other MI/DCI ratios were less effective or even exerted negative effects on the clinical-pathological conditions.


The formula with high DCI content demonstrated to be unfavorable, worsening PCOS pathological features, in line with the ovarian DCI paradox .


Moreover, Ravanos and colleagues tested whether different MI and DCI concentration in follicular fluids could correlate with blastocyst quality . They showed that high levels of DCI in follicular fluid of healthy young women impaired their blastocyst quality, meanwhile lower D-chiro-inositol content correlated with a good blastocyst quality and a satisfying implantation rate and pregnancy rate. Thus, for oocyte and blastocyst evaluation, we can state that MI and DCI are, respectively, “high quality” and “low quality” biomarkers.


A recent metaanalysis evaluated the efficacy of treatments with MI, alone or combined with DCI in a 40:1 ratio for 12–24 weeks, in nine randomized controlled trials comprising 247 cases and 249 controls . The authors considered fasting insulin concentrations as primary outcome, and HOMA-IR index, testosterone, androstenedione, and sex hormone-binding globulin (SHBG) plasma levels as secondary. Significant reductions in fasting insulin and HOMA-IR index were found after inositol supplementation, suggesting a slight trend toward testosterone decrease was observed with respect to controls, whereas androstenedione levels remained unchanged.


Definitely, MI was able to significantly increase SHBG levels only after at least 24 weeks of administration. Concerning the androgenic hormones, the different effects obtained on androstenedione and testosterone levels should be further investigated.


The authors of this metaanalysis recommended to avoid exclusive DCI supplementation essentially for three reasons: high doses of DCI can be detrimental for ovaries and oocyte maturation; DCI cannot be converted into MI, losing the action of the latter; MI deficiency is correlated with many conditions of insulin resistance. Therefore, the metaanalysis strongly supported MI supplementation to improve the metabolic profile of PCOS patients.


A study was carried out to evaluate the improvements of clinical and body composition in 43 overweight and obese PCOS patients .


The patients were randomly divided into 3 groups and treated for 6 months: group 1 with 1200 Kcal diet only; group 2 with diet plus MI (2 g MI and 200 μg folic acid in powder, twice daily); group 3 with diet associated to MI and DCI in the 40:1 ratio (2 soft gel capsules, containing 550 mg MI, 13.8 mg DCI, and 200 μg folic acid, per day).


At the end of the treatment, weight, BMI, waist, and hip circumferences decreased significantly in all the patients. The addition of MI plus DCI to the diet seems to accelerate the diminution of weight and fat mass, with a slight increase of lean mass. Thus, the addition of MI plus DCI in the 40:1 ratio to the diet contributed to improve at most the restoration of regularity of the menstrual cycle.


With the aim to provide a clinical confirmation to the research by Bevilacqua , Nordio and colleagues compared some PCOS treatments with different MI/DCI ratios (DCI alone, and in 1:3.5; 2.5:1; 5:1; 20:1; 40:1, 80:1). They randomly allocated 56 women into 7 groups treated with in total, 2 g of inositols twice daily for 3 months. Ovulation was the primary outcome, whereas the secondary outcomes included BMI, menses, basal and postprandial insulin levels, HOMA-IR index, FSH, LH, SHBG, 17-β-estradiol, free testosterone. Among the seven tested formulations, again the 40:1 ratio achieved the best results, restoring ovulation in 62.5% of women.


Therefore, these results confirmed the study carried out with the PCOS mice . The authors highlighted that the two stereoisomers, though their almost same chemical structure, in some cases demonstrate a different clinical efficacy. In conclusion, the 40:1 MI/DCI ratio is the supplementing approach with the better expectation, as also supported by some clinical experiences.


Inositol-resistance


In the 35% of PCOS women inositols failed to significantly improve the metabolic and hormonal parameters and restore ovulation.


This therapeutic inefficacy of inositols in some patients is called “inositol-resistance” .


It is likely that such a problem derives from a reduced or absent absorption of inositol due to unclear or unpredictable conditions (obesity, chronic intestinal diseases, dysbiosis). To increase the absorption, MI was combined with the whey protein alpha-lactalbumin (alpha-LA), an excellent “carrier” for metal ions (especially Ca 2 + and Fe 2 + ) and for vitamin D .


Alpha-LA may act as an enhancer of passage through biological barriers. Monastra and co-workers observed in vivo and in vitro an increased MI passage in the presence of alpha-LA through the human intestinal Caco-2 cell monolayer, commonly used as in vitro model of gut mucosa . At first, they observed on healthy volunteers that MI bioavailability was modified by the concomitant administration of alpha-LA; indeed, the administration of alpha-LA with MI in a single dose, significantly increased the plasma concentrations of MI compared to when administered alone. Secondly, MI absorption in Caco-2 cells was improved in the presence of digested alpha-LA, and this change was associated with an increase in tight junction permeability .


A subsequent study from Montanino Oliva et al. on PCOS patients treated with MI plus alpha-LA confirmed the clinical efficacy of this formulation . This therapy drastically reduced the number of inositol resistant patients, with significant progress, promising a way to overcome some current limitations of inositol therapy.


Hormone D (vitamin D)


“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.


Alpha-lipoic acid


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 .


Omega 3


Among dietary factors, omega 3 fatty acids especially marine n-3 PUFA (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) have antiinflammatory, antiobesity and antiinsulin resistance functions . Omega 3 fatty acids can improve insulin sensitivity by decreasing the production of inflammatory cytokines, including TNFα, IL-6 and increasing secretion of antiinflammatory adiponectin .


Several studies evaluated the effect of omega 3 fatty acids in women with PCOS, however, referring to different findings. Results of a metaanalysis of randomized controlled trials reported that supplementation with omega 3 fatty acids may not have a beneficial effect on improving insulin resistance in women with PCOS . On the other hand, other randomized controlled clinical trials showed that omega 3 fatty acids may contribute to the improvement of metabolic complications and had some beneficial effects on serum adiponectin levels, insulin resistance, and lipid profile in patients with PCOS .


The study from Khani and co-workers showed that a 6-month treatment with omega 3 in women with PCOS improved the values of waist circumference, HDL, LDL, triglycerides, and regularity of periods in comparison to control group . However, no significant changes were observed in other parameters such as weight, number of ovarian follicles, size of ovary, bleeding volume, menstrual bleeding, and hirsutism score between intervention and control groups.


These results were similar to Nadjarzadeh’s study , that showed how after 8-week treatment with omega 3, the percentage of regular menstruation in the treated group was significantly higher than the placebo group, likely due to the decrease in testosterone concentration.


Interestingly, vitamin D and omega 3 fatty acid co-administration for 12 weeks had beneficial effects on mental health status, total testosterone level, serum hs-CRP, plasma total antioxidant capacity and malondialdehyde levels, and gene expression of IL-1 and VEGF among women suffering from PCOS, supporting the antiinflammatory and immunomodulatory roles of combined vitamin D and omega 3 supplementation .


Melatonin


Melatonin ( N -acetyl-5-methoxytrypamine) is an indolamine hormone, regulated by photoperiod. In detail, its production and secretion are promoted in response to darkness meanwhile light suppresses the secretion .


Several organs produce melatonin, such as gastrointestinal tract, skin, retina, bone marrow, and lymphocytes . Moreover, the female reproductive organ, including the follicular cells, oocytes, and cytotrophoblasts are considered melatonin sources .


Also, melatonin is a potent free radical scavenger with protective effects in female reproductive organs; for instance, it is involved in the protection of the oocytes against oxidative stress, particularly at the time of ovulation.


Melatonin levels in serum and follicular fluid of PCOS patients are different from healthy women. In PCOS patients, melatonin level in serum is usually higher than in healthy women, which is considered as a sign of diagnosing PCOS . However, in the follicular fluid a reverse condition occurs. Due to fewer uptake of melatonin in ovarian follicle in PCOS patients, follicular fluid contains lesser melatonin compared to the healthy condition .


Melatonin seems to promote follicular maturation and ovulation through the protection of follicles against oxidative stress and their rescue form atresia . Furthermore, melatonin showed protective effects on corpus luteum against ROS via its antioxidant effects . Since melatonin level in follicular fluid of PCOS women is notably lower than in healthy women, the ovulation can be highly affected.


It has been reported that melatonin administration can compensate the reduction of this hormone in follicular fluid and can overcome ovulation problems . Melatonin treatment indicated protective effects against metabolic and reproductive abnormalities in PCOS patients. Indeed, it may reduce peripheral tissue sensitivity to insulin , and its administration in PCOS patients significantly affects body characteristics, including reduced body weight, body mass index and intra-abdominal fat .


During the ovulatory process, ROS are produced within the follicles; for this reason, the scavenging activity of melatonin plays an important role during ovulation.


The ROS generated from mononuclear cells are elevated in women with PCOS , significantly increasing serum lipid peroxidation leading to a poor oocyte quality .


Melatonin reduces oxidative stress and causes oocyte maturation and luteinization of granulosa cells, making it as an effective treatment for PCOS patients. Intra-follicular melatonin concentration was considerably lower in PCOS patients giving rise to anovulation and poor oocyte quality in these patients. The supplementation of melatonin alone or in association with other compounds in PCOS women has been shown to increase the intrafollicular melatonin concentration, reduce the intrafollicular oxidative stress, and increase the fertilization and pregnancy rates.


Melatonin also improves the production of progesterone from corpus luteum in PCOS patients. The deficiency of melatonin alters gonadotrophin secretion, reduces the synthesis of FSH, and increases the synthesis of LH, the latter being the major change detected in PCOS patients .


It has been demonstrated that melatonin administration improves oocyte quality ; furthermore, after assumption, it is able to accumulate in the follicular fluid, where it reduces intrafollicular oxidative damage and elevates fertilization and pregnancy rates.


Interestingly, several clinical trials have demonstrated that supplementing an association of melatonin and myo-inositol oocyte quality and IVF outcomes improved in both PCOS patients and normal subjects .


The trial conducted by Pacchiarotti and colleagues showed on a significant number of patients ( n = 526) the synergistic action between melatonin and myo-inositol, positively influencing the quality of oocytes and embryos.


This was a crucial achievement also for enhancing the outcomes in IVF when it is carried out in PCOS women.


Probiotics


Dysbiosis of gut microbiota may be a potential pathogenetic factor for PCOS development. In this context, modification of gut microbiota with probiotic, prebiotic, and symbiotic agents suggest that these products may serve as new treatment options for PCOS.


Probiotics are living microbial dietary supplements found in dairy products and having synergism with the gut microbiota . Studies demonstrated their beneficial effects on metabolism, especially under inflammatory conditions ; also, their consumption improves fasting blood glucose and antioxidant status in patients with type 2 diabetes .


Both chronic state of inflammation and IR, involved into the etiology of PCOS, are associated with the dysbiosis of gut microbiota (DOGMA theory).


The background of DOGMA involves an imbalance in gut microbiota, namely increasing the transition of Gram-negative bacteria into systemic circulation, inducing a chronic inflammatory response in the host. This inflammatory process affects insulin receptor function and PCOS-associated pathways such as androgen biosynthesis. Therefore, to overcome the pathophysiologic conditions of PCOS, probiotic supplements are recommended .


Yadav and colleagues showed that a probiotic supplemented diet delayed the onset of glucose intolerance, hyperglycemia, hyperinsulinemia, and dyslipidemia in diabetic rats . He and colleagues demonstrated that lactic acid bacteria alleviated PCOS in a rat model by regulating sex hormone-related gut microbiota.


Shoaei and co-workers investigated the effects of an 8-week probiotic supplementation on pancreatic β cells and C-reactive protein (CRP) in patients with PCOS. They observed a reduction in fasting blood sugar and serum insulin levels, without CRP levels significantly change .


A recent systematic review and metaanalysis evaluated the effectiveness of probiotics on metabolic, hormonal, and inflammatory parameters of PCOS . The authors observed that probiotic administration was associated with a significant improvement of HOMA-IR, BMI, Ferriman-Gallwey, serum triglycerides, serum testosterone, hs-CRP, and other parameters .


Conclusions


Several studies highly supported the 40:1 MI/DCI ratio as the best choice for PCOS treatment in order to restore ovulation in these patients, demonstrating that DCI activity is beneficial mainly in a specific balance with MI, whereas the progressive increase in the concentration of DCI causes the parallel loss of the beneficial effects at the reproductive level with concomitant blastocyst quality.


Emerging data also support alternative compounds, alone or in combination with the aforementioned strategies, with favorable effects on ovulation, insulin resistance and inflammation. Nevertheless, additional studies are required, in order to assess the effectiveness of these supplements in preventing negative impacts of PCOS features.



References

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Nov 27, 2021 | Posted by in GYNECOLOGY | Comments Off on Inositols and other supplements in the management of PCOS

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