A Clinical Guide to Inositols 0323916732, 9780323916738

A growing body of research demonstrates the potential benefits of the administration of inositol isomers in the treatmen

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A Clinical Guide to Inositols
 0323916732, 9780323916738

Table of contents :
A Clinical Guide to Inositols
Introduction to the history of inositols: A tale of scientists
Inositol: Early characterization in plants
Inositol in animals: From metabolism to its pivotal roles in signaling processes
Inositol in clinical practice: Early findings and stereoisomers
Physiological and pathophysiological roles of inositols
Evolution of inositols
Activity of myo-Ins and its stereoisomers
Glycemic control
Human reproduction
myo-Ins-derived phospholipids
Natural sources of inositols
Inositol endogenous synthesis and catabolism
Inositol transport system
Inositols levels in plasma and tissues
Inositols: From the physiology to the pathophysiology
Effectiveness of Myo- and d-chiro-inositol in the treatment of metabolic disorders
The global ``metabolic epidemic´´: Common soil for a clustering of disorders
Prevalence and clinical manifestations
Pathophysiological components of metabolic disease
Energy homeostasis and glucose metabolism: The emerging role of inositols
Inositols are physiologically involved in insulin signaling
Inositols are involved in insulin secretion by pancreatic β cells
Molecular mechanisms and metabolic effects: Experimental and animal data
Myo-inositol (MI)
d-Chiro-inositol (DCI)
d-Pinitol (DPI)
Translating the pathogenetic background into a putative therapeutic role of inositols in metabolic disease
Targeting obesity
Targeting insulin resistance and type 2 diabetes mellitus
Targeting other aspects of metabolic syndrome
Treating PCOS with inositols: Choosing the most appropriate myo- to d-chiro-inositol ratio
Introduction: PCOS and polycystic ovaries
Mechanisms of action of inositol and its role in PCOS
Inositols and glucose metabolism
Inositols and steroidogenesis
Clinical and preclinical data
Inositols in PCOS treatment
The 40:1 approach
High d-chiro-inositol dosage
Intestinal absorption of inositols
Overcoming inositol resistance
Effect of inositols on glucose homeostasis in PCOS
Effect of inositols on ovarian function in PCOS
Inositol resistance
Myo-inositol could restore peripheral inositol depletion induced by treatments for psychiatric and neurologica ...
Side effects of the pharmacological therapies: Consequences of peripheral inositol depletion
The efficacy of inositol supplementation
Effectiveness and safety of inositols: A matter of dosages and ratios
The role of inositols in maternal adaptation, placental function, and regulation of fetal growth and development
Maternal adaptation to pregnancy
Insulin resistance and gestational diabetes
Cardiovascular system and preeclampsia
Lipid metabolism and mobilization
Placenta as a regulator of nutrient/waste exchange and maternal-fetal communication
The placenta as a regulator of maternal-fetal exchange of nutrition and waste
Circulation in the placenta
Oxygen and carbon dioxide diffusion across the placental barrier
Transport of nutrients across the placenta
Glucose transport
Amino acid transport
Fatty acid transport
Na, K, Cl, and calcium transport
The placenta as a mediator of maternal-fetal communications through extracellular vesicles
Metabolism and passage of inositol across the maternal-placental-fetal axis
Inositols in the regulation of fetal growth and development
Neural tube defects
Fetal lung maturation and neonatal respiratory distress
Fetal growth and size at birth
Altered placental inositols in GDM and preeclampsia could impact fetal growth
Inositol and the prevention of adverse fetal outcomes, with particular reference to neural tube defects
Neural tube defects (NTDs)
Types of NTD
Causes of NTDs
Primary prevention of NTDs-Folic acid (FA) supplementation
Inositol and prevention of NTDs
Counseling families at risk of NTD recurrence
Conclusions and future directions
Inositol supplementation for preventing gestational diabetes mellitus
Gestational diabetes mellitus (GDM) around the World: Basics and problematics
Classification and definition
Diabetes in pregnancy
Gestational diabetes mellitus (GDM)
Diagnosing GDM
Diagnostic criteria
GDM-related complications
Maternal complications
Fetal and neonatal complications
Fetal programming and childhood complications
Long-term maternal effects
Prevention and treatment of GDM with inositol
The role of inositol in insulin signaling
Current management of GDM
Nutritional supplementation for preventing and/or treating GDM
Inositols for GDM prevention
Inositols for GDM treatment
Use of myo-inositol in the treatment of PCOS symptoms in adolescents
Prevalence and etiology
Diagnostic challenges
Clinical manifestations and consequences
Pharmacological therapies
PCOS and myo-inositol
Treatments with inositols in IVF procedures
Inositols in the male reproductive process
Male treatment
Inositols in the female reproductive process
PCOS metabolism and ovulation: MI and DCI supplementation
Anovulation and DCI
Inositols and assisted reproduction technologies (ART)
Supplementation with d-chiro-inositol in women
Inositol properties
DCI role in the hormonal signaling pathway
DCI supplementation
DCI properties
Uterine myomas
Endometrial hyperplasia
Breast and endometrial tumors
Mood disorder
Ovarian hyperstimulation syndrome (OHSS)
The ovarian paradox
Application of myo-inositol and d-chiro-inositol in andrological issues
Role of myo-inositol in male fertility
Role of d-chiro-inositol in male fertility
Role of oxidative stress and antioxidant treatments
The antioxidant system in the male fertility
Antioxidant supplementation for male fertility
Myo-inositol for subclinical hypothyroidism and potential prevention of thyroid tumors
Myo-inositol and hypothyroidism
Myo-inositol and subclinical hypothyroidism in pregnancy
Myo-inositol in thyroid tumorigenesis
Inositols as adjuvant treatments in oncology
Introduction (how it all began)
Epidemiological clues
Inositol and cancer: Basic mechanisms
Antioxidant effects
Modulation of immune system
Inositol hexakisphosphate (InsP6): The cumulated evidence
Inositol and cancer types
Colorectal cancers
Breast cancer
Lung cancers
Hematological cancers
Brain tumor
Endocrine cancers
Thyroid cancer
Neuroendocrine neoplasms
Adrenal tumor
Role of insulin resistance in endocrine tumors
Chemopreventive and therapeutic efficacy of InsP6 in animal studies
Clinical trials on inositol in human cancers
Colorectal cancer
Breast cancer
Lung cancer
Perspectives and outstanding issues

Citation preview



Edited by VITTORIO UNFER Systems Biology Group Lab, Rome, Italy

DIDIER DEWAILLY Endocrinology, Reproductive Medicine, Lille University Hospital, Lille, France

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-91673-8 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Patricia M. Osborn Editorial Project Manager: Timothy J. Bennett Production Project Manager: Fahmida Sultana Cover Designer: Greg Harris Typeset by STRAIVE, India


Marialuisa Appetecchia Oncological Endocrinology Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy Cesare Aragona Systems Biology Group Lab, Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Daniele Barbaro U.O. Endocrinology ASL Nord Ovest, Tuscany, Italy Salvatore Benvenga Department of Clinical and Experimental Medicine, University of Messina; Interdepartmental Program of Molecular & Clinical Endocrinology and Women’s Endocrine Health, University Hospital, Messina, Italy Arturo Bevilacqua Department of Dynamic, Clinical Psychology and Health Studies, Sapienza University; The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy Mariano Bizzarri The Experts Group on Inositol in Basic and Clinical Research (EGOI); Systems Biology Group Lab; Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Tonino Cantelmi Institute for Interpersonal Cognitive Therapy; The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy Pietro Cavalli Department of Medical Genetics, Humanitas Research Hospital, Milano, Italy Shiao-yng Chan Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore; Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Singapore Andrew J. Copp Developmental Biology & Cancer Research & Teaching Department, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom Rosario D’Anna Department of Human Pathology, University of Messina, Messina, Italy Didier Dewailly The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy; Faculty of Medicine, University of Lille/Institut National de la Sante et de la Recherche Medicale (INSERM) Laboratory of Development and Plasticity of the Neuroendocrine Brain, Jean-Pierre Aubert Research Centre, Lille, France




Cherubino Di Lorenzo The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome; Department of Medico-Surgical Sciences and Biotechnologies, La Sapienza University Polo Pontino, Latina, Italy Evanthia Diamanti-Kandarakis Department of Endocrinology and Diabetes, Professor of Endocrinology, Hygeia Hospital, Athens, Greece Simona Dinicola The Experts Group on Inositol in Basic and Clinical Research (EGOI); Systems Biology Group Lab, Rome, Italy Maria Salome` Bezerra Espinola Systems Biology Group Lab, Rome, Italy Fabio Facchinetti UOC Gynecology and Obstetrics, Mother-Infant Department, University of Modena, Modena, Italy Nicholas D.E. Greene Developmental Biology & Cancer Research & Teaching Department, Great Ormond Street Institute of Child Health, University College London, London, United Kingdom Moshe Hod Mor Comprehensive Women’s Health Care Center, Tel-Aviv, Israel; FIGO, Pregnancy and Non-Communicable Diseases Committee, London, United Kingdom Zdravko Kamenov Clinic of Endocrinology, Alexandrovska University Hospital, Medical University of Sofia, Sofia, Bulgaria Antonio Simone Lagana` Unit of Gynecologic Oncology, ARNAS “Civico – Di Cristina – Benfratelli”, Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (PROMISE), University of Palermo, Palermo, Italy Giovanni Monastra Systems Biology Group Lab; The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy John E. Nestler The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy; Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States Maurizio Nordio Department of Experimental Medicine, University “Sapienza”, Rome, Italy Mario Montanino Oliva Altamedica Reproductive Medicine, Unicamillus Medical University, Rome, Italy


€ Ali Cenk Ozay Department of Obstetrics and Gynecology, Near East University, Research Center of Experimental Health Sciences, Near East University Hospital, Nicosia, Cyprus Olga Papalou Department of Endocrinology and Diabetes, Hygeia Hospital, Athens, Greece Lali Pkhaladze The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy; Zhordania and Khomasuridze Institute of Reproductology, Tbilisi, Georgia Giuseppina Porcaro Women’s Health Centre, Terni, Italy Nikos Prapas Third Department of OB-GYNAE, Aristotle University of Thessaloniki; IVF Laboratory, IAKENTRO Fertility Centre, Thessaloniki, Greece Scott Roseff South Florida Institute for Reproductive Medicine—IVFMD, Boca Raton; Clinical Assistant Professor, Reproductive Endocrinology & Infertility, Department of Clinical Sciences, Division of Obstetrics and Gynecology, Dr. Kiran C. Patel College of Allopathic Medicine-Nova Southeastern University MD, Ft. Lauderdale, FL, United States Christophe O. Soulage University of Lyon, CarMen Lab, INSERM 1060, INRAE U1397; Universite Claude Bernard Lyon 1, Lyon, France Annarita Stringaro National Center for Drug Research and Evaluation, Italian National Institute of Health, Rome, Italy Vittorio Unfer The Experts Group on Inositol in Basic and Clinical Research (EGOI); Systems Biology Group Lab, Rome, Italy Monica Vazquez-Levin Institute of Biology and Experimental Medicine (BYME, CONICET-FIBYME), National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina Ivana Vucenik Department of Medical and Research Technology; Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, United States Artur Wdowiak Chair of Obstetrics and Gynecology, Faculty of Health Sciences, Medical University of Lublin, Lublin, Poland Tony Chiu Tak Yu IVF Centre, Hong Kong, China



Dedicated to the memory of Professor Joseph Larner, whose findings contributed to the current research on inositols and inspired the birth and activities of EGOI.


If this book had seen the light as Vittorio and I first conceived it, the reader would have turned these pages to find an exhaustive—yet quite disconnected—list of clinical applications of inositol-based treatments. However, because the research on the properties and clinical effects of inositols has made huge steps forward over the most recent years, we realized that our mission was far more complex than originally envisaged. As first task, we felt the need to dismantle the ingrained perception that inositol implicitly refers only to myo-inositol. Surprising fact (that not everyone knows), it all actually started with D-chiro-inositol. Indeed, the identification in 1988 of an insulin, second messenger, based on D-chiro-inositol prompted a great interest toward its biological role in the following years. It is hard to pinpoint the reasons why such interest slowly faded and was left aside for more than 20 years, only to resurface recently. Thanks to accumulating new evidence, we have now defined that myo-inositol and D-chiro-inositol possess very distinct and specific physiological activities and that both isomers are essential for cell physiology. Clearly, our understanding of the properties of myo-inositol has grown particularly quickly, as it is the most abundant among the natural inositol isomers and is present in all tissues and organs of the human body. Its role in improving the sensitivity toward insulin and FSH is quite well established, and myo-inositol is currently an effective asset for managing the symptoms of the polycystic ovary syndrome, especially when this is associated with insulin resistance. Still novel and exciting clinical applications of myo-inositol are emerging, including the management of metabolic syndrome and prevention of gestational diabetes mellitus and neural tube defects, but also interesting applications in the fields of cardiology and psychiatry. More recently, myo-inositol gained an interest for oncological studies, as its phosphate derivatives display epigenetic activity in chromatin remodeling and DNA methylation, exerting pivotal functions during morphogenesis and cell fate commitment. On the other hand, the study of D-chiro-inositol properties remains a largely uncharted territory, and future investigations will be critical to delineate the use of D-chiro-inositol in clinical practice. Based on the latest reports, while both isomers are second messengers of insulin, D-chiro-inositol seems to be heavily involved in the biosynthesis of androgens. Moreover, given its physiological concentration range, D-chiro-inositol seems an extremely potent effector with respect to myo-inositol, and this characteristic should be considered when defining supplementation regimens. As myo-inositol and D-chiro-inositol physiologically coexist, it is becoming increasingly evident that the ratio of the two isomers determines some of the cellular properties




and functions. Such ratio is maintained by an insulin-dependent enzyme that converts myo-inositol into D-chiro-inositol, and conditions that alter Insulin levels may disrupt the physiological inositol balance, thus leading to pathological manifestations. Our understanding of physiological role of myo-inositol and D-chiro-inositol is paramount to devise a tailored supplementation with either isomers, or the proper combination of the two. Overall, we know that inositols participate in a wide number of complex physiological pathways, and the findings from current research keep feeding us constantly with insights on the properties of myo-inositol and D-chiro-inositol. With this book, Vittorio and I hope to convince our readers that it is high time to begin talking about inositols—rather than inositol—bearing in mind that we know only some of the biological roles of the two isomers. We also mean to make physicians and researchers aware that inositol-based treatments need to be tailored on the characteristics of patients, and not exclusively on the pathological conditions. Fostering research on inositols is necessary to uncover the full clinical application range of supplementation approaches, and The Experts Group on Inositol in Basic and Clinical Research was established in early 2020 precisely with this purpose. The group brings together more than 40 international scientists and clinicians with outstanding experience in the field, promoting collaborations for scientific research and, most of all, sparking discussion on novel clinical applications of inositol-based supplementation regimens. With an active outreach program, a published position paper, and already two congresses, EGOI stands out as the leading voice for inositol research. All the chapters featured in this book are contributions by EGOI members, who enthusiastically took part in creating this updated overview of the current research areas that encompass investigations on the properties and applications of inositols. Didier Dewailly


Our special thanks go to Dr. Gianpiero Forte, Mr. Riccardo Gambioli, Dr. Michele Russo, and Dr. Elisa Lepore for their contribution in revising and editing the chapters of this book.



Introduction to the history of inositols: A tale of scientists Vittorio Unfera,b and John E. Nestlera,c a

The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy Systems Biology Group Lab, Rome, Italy c Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, United States b

Inositol: Early characterization in plants Inositol is a naturally occurring cyclic polyol. Its discovery dates back to mid-1800s by Scherer, who isolated the compound from muscle tissue. Scherer named the substance “myo-inosite”, which in chemical language indicated a polyalcohol carbohydrate detectable in muscular fibers [1]. Years later, Maquenne extracted myo-inositol from leaves, then determined its molecular weight and its structure, and observed the absence of any reducing activity. Subsequently, he purified the compound from horse urine, demonstrating that inositol is present both in plants and in animals [2–4]. In 1919, Posternak isolated and characterized phytic acid from leaves. He found out that this compound is indeed the hexa-phosphate of myo-inositol, suggesting that inositol undergoes chemical reactions in the plants [5]. Years later, Needham standardized the purification procedures, allowing quantifications and selective isolations [6]. Posternak later determined the structure of two different inositol isomers, myo- and scyllo-inositol, discovering that natural inositol makes up a family of isomers [7]. On these bases, future investigations shed light on some of the biological features of inositols, including conversion between isomers and ratios in tissues and organs. Many of the roles of inositols in vivo are currently under investigation, and inositol physiology still represents a hot topic of research.

Inositol in animals: From metabolism to its pivotal roles in signaling processes After optimizing the method for the purification of inositol, Needham focused his research on the physiology of inositol in animals. Through a series of elegant experiments, he demonstrated indeed that animal do synthesize inositol, independently of dietary intake. As a first step, Needham determined the content of inositol in hen’s eggs, both unincubated and at the end of development. He found out that inositol concentration increased ten times in the period that goes from preincubation to hatching, A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00014-5

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A clinical guide to inositols

concluding that an endogenous source of inositol does exist. Such source may consist either in the release of inositol phosphates from the membranes or in a de novo endogenous biosynthesis or, virtually, in both these processes. To shed light on this mechanistic puzzle, Needham designed an in vivo experiment with rats. The animals were fed with a high-salt, cyclose-free diet for eight months, in order to increase urinary excretion and deprive them of exogenous cycloses. At the end of the experiment, Needham recorded inositol excreted in the urine, concluding that animals were able to synthesize it de novo, also in a regimen of exogenous deprivation [8]. Given these findings, two main questions were still open at that time, engaging scientists in speculations and hypothesis. Which are the physiological roles of inositol? Where is inositol localized in animal organisms? Fast-forward almost twenty years, Folch was studying cephalin, a membrane phospholipid of neurons, in cow’s brain and the role of brain lipids. During his experiments, he serendipitously found out that myo-inositol-based phosphates are constituents of structural lipids, providing the first insight into their crucial role in plasma membranes, especially in the brain [9]. A few years later, the Hokins (husband and wife) discovered that inositol is not only a structural constituent of membranes, but also an active molecule in biological signaling. Indeed, they found that neurotransmitters, such as acetylcholine, induce cytosolic enrichment with inositol phosphates in the post-synaptic neurons. This finding led the Hokins to hypothesize that inositol undergoes release from the membranes and subsequently participates in intracellular signaling processes. They defined this sequence of events as “PI effect” [10]. The research that Ballou and Dawson carried out in the following decade added new elements to Folch’s and Hokins’ findings. Specifically, Dawson first identified and purified the phospholipid phosphatidylinositol 4,5 bisphosphate, which he called “tri-phosphoinositide” [11], while Ballou discovered that multiple myo-inositol phosphates (including mono-, di-, and triphosphate) exist in the same tissue [12]. Years later, during the period of excitement that followed the discovery of cyclic AMP and the mechanism of second messengers, Berridge was working on calcium mobilization in blowfly salivary glands. Indeed, he was a physiologist who crossed the path of molecular biology and biochemistry, and at that time, he was studying the mechanisms that prompt the secretion of saliva through the activation of membrane ionic channels [13]. Riding the wave of enthusiasm for cyclic AMP, he tested whether this popular molecule could be responsible for the actions he observed during the activation of membrane ionic channels. Indeed, as he reported, he felt excited when he found that cyclic AMP was responsible for the potassium ion uptake, but he also felt puzzled when he observed it had no effects at all to chlorine, which Berridge knew to regulate secretion [14,15]. In those years, findings on cyclic AMP drained all the attentions of scientists, fascinated by the brand-new concept of second messengers, which unfortunately represented an exclusive name for cyclic AMP. Indeed, the greater part of scientists believed that cyclic AMP

Introduction to the history of inositols

was the only second messenger molecule, and they almost ignored data indicating the possible existence of other second messengers. Hopefully pursuing his objectives, Berridge found literature evidence suggesting that Ca2+ could represent the effector of saliva secretion. Then, he tested if Ca2+ was responsible for the opening of chlorine channels. Performing his experiments, in the absence or in the presence of Ca2+, revealed that, as he expected, Ca2+ rather than cyclic AMP was the real effector of chlorine uptake [16]. At this point, Berridge research crossed the work of Michell, who strongly believed that the PI effect detected by the Hokins was further propagated by Ca2+ [17]. Berridge then started analyzing inositol in membranes, until he demonstrated that an increase in inositol release from membranes leads to higher Ca2+ levels in the gland. This was the first demonstration that a compound different from cyclic AMP could regulate extracellular-tointracellular communications [18]. At this point, Berridge only had to identify the inositol species involved in Ca2+ mobilization. He was lucky enough to work “a few miles down the road” from Dawson’s lab, one of the only two laboratories in the world that could provide inositol phosphate standards at that time. Concurrently with Berridge’s research, Irvine—working at Dawson’s lab—was trying to identify the real effector of the PI effect. Indeed, he was a biochemist with a great expertise in inositides, as his main research topics included inositol derivative formation, with a special focus on the kinetics of the processes. While Berridge worked on blowfly glands, he carried his experiments on platelets, obtaining poor results as these cells display poor inositol uptake. Like Berridge during his own research, Irvine was inspired by Michell, who persistently believed that the real effector of PI effect was phosphatidylinositol 4,5-bisphosphate, a hydrophilic inositol-based head of membrane lipids. During the collaboration between Berridge and Irvine, they struggled to find the optimal cell model in which they could carry their experiments. In fact, the poor inositol uptake of platelets and the difficulty of analyzing salivary gland cells had left them with few or null ideas on how to proceed. Luckily, Berridge attended a meeting in Amsterdam where he was invited as a speaker. Sch€ ulz, the person who spoke right after him, made her report about permeabilized pancreatic cells, which Berridge guessed as the optimal model for his and Irvine’s experiments [19,20]. Working on pancreatic cells was troublesome at the beginning, but the intervention of Irvine, who was able to prepare high-quality standards, led to the final demonstration they were looking for. Indeed, they observed that phosphatidylinositol 4,5-bisphosphate is cleaved to form the water-soluble inositol 1,4,5-trisphosphate, and the latter mobilizes Ca2+ from the endoplasmic reticulum, activating calcium downstream effectors [21]. They demonstrated this mechanism in pancreatic cells, but the magnitude of their finding prompted the quick research on similar inositol activities in other cellular systems. The collaboration of these keenminded academics thus first clarified inositol role in signaling. Through their knowledge in biochemistry and molecular biology, they identified inositol trisphosphate, which is today considered as one of the most important second messengers [22]. Given the huge



A clinical guide to inositols

amount of data on inositol available today, one may take this finding for granted, but it is probably the most important discovery ever made on inositol, defining its pivotal role in physiology. After the discovery of inositol’s signaling role, further evidence started emerging on this topic. While Irvine and Berridge were clarifying the role of inositol phosphates, other scientists were working on other signaling mechanisms, especially insulin. The work of Larner already focused on insulin signal mediators, and its group had already isolated the compounds believed to be involved in such signaling [23]. Despite this, it was Saltiel who first highlighted that such insulin mediators were indeed “derived from an inositol glycolipid” [24,25]. Along with this finding, these scientists started noting differences in insulin mediators. The group of Larner isolated two different mediators of insulin signal, which they found to be characterized by diverse activities. The first mediator activates pyruvate dehydrogenase phosphatase, explaining some of the intracellular actions of insulin, while the second mediator had a pivotal role in the inhibition of protein kinase A. Later, Larner’s group characterized these two mediators, revealing that they are indeed inositol phosphoglycans [26]. The first one, inhibiting pyruvate dehydrogenase phosphatase, is composed by galactosamine and D-chiro-inositol [27]. The second one, the inhibitor of protein kinase A, is made out of myo-inositol, glucosamine, galactose, and ethanolamine [28]. Thus, for the first time, inositol stereoisomers were noted to have different activities, an evidence that will further open several debates. Through an elegant experiment with radiolabeled inositol, Larner discovered the tissue-specific conversion rates of stereoisomers [29]. Hence, Larner and colleagues identified D-chiro-inositol deficiency as a common feature of diabetic patients and observed that insulin prompts the biosynthesis of D-chiro-inositol-containing phospholipids [30]. Concomitantly, further studies by Larner in diabetic rhesus monkeys and diabetic rats pointed out the clinical potential of D-chiro-inositol as an insulin-mimetic/insulin sensitizer compound [31]. During those years, Nestler was investigating insulin effects in gynecological and hormonal contexts. He studied lipoproteins and hormones, focusing on their relations with insulin. One of the most important intuition Nestler had concerned the correlation between insulin-like growth factor I (IGF-1) and women hyperandrogenism, which he found studying the regulation of ovarian steroidogenesis [32]. He subsequently observed a similar effect through the action of insulin, finding a direct relation between hyperinsulinemia and hyperandrogenism [33]. Moreover, he found a reduction in ovarian production of estrogens following insulin treatment [34]. Working in collaboration, Larner and Nestler identified inositol glycans as mediators of the signal from insulin and related molecules that impact steroidogenesis [35]. Nestler and colleagues then demonstrated undoubtedly that the D-chiro-inositol-containing phosphoglycans transmitted the signal of insulin, leading to testosterone accumulation in the ovaries [36]. While Nestler was investigating the role of D-chiro-inositol in steroidogenesis, Chiu was approaching studies on myo-inositol. Based on the relation between myo-inositol

Introduction to the history of inositols

deficiency and diabetic embryopathies, he decided to investigate whether myo-inositol could affect embryo development or not. Due to its work on in vitro fertilization (IVF), he used the sera of women undergoing IVF as the primary source of myo-inositol. He noted that sera from patients with good IVF outcomes, when added to culture media, supported a better development of mouse embryos in vitro, as they contained higher levels of myo-inositol. Nonetheless, sera of women who had abortions, although supporting embryo development as well, displayed lower quantities of myo-inositol. Moreover, he observed that in some cases, the embryo development improved after the supplementation of myo-inositol [37]. These findings prompted other research to investigate whether the relevant effects of myo-inositol were due to the content in serum or in follicular fluid. Chiu’s main findings concerned the follicular fluid, which he found to impact the oocyte quality depending on the myo-inositol content. Indeed, myo-inositol content was also directly related to estradiol content in follicular fluid. Then, he proposed, and later confirmed, that myo-inositol content in follicular fluid should be considered as a marker of good oocyte quality [37].

Inositol in clinical practice: Early findings and stereoisomers Growing evidence on the roles of inositols in physiology provided a solid rationale for testing the potential of such molecule in clinical studies. Indeed, the first clinical trials involving myo-inositol treatments had been already carried out during early 1950s by Dotti and Felch. They had pointed out the positive effect of inositol on cholesterol levels in hypercholesteremic infarct survivors and in diabetics, but the new evidence opened further scenarios [38,39]. Nestler decided to test D-chiro-inositol as an insulin-sensitizing treatment to induce ovulation in women with PCOS by removing the excess insulin stimulus on the ovary. As expectable, he demonstrated that D-chiro-inositol induced ovulation in obese, insulin-resistant women with PCOS by reducing insulinoverburdening stimulus [40]. Years later, Nestler and colleagues confirmed these promising results on ovulation induction also in lean insulin-resistant patients with PCOS [41]. The clinical research on inositol yielded great results during the following years until 2008, when the group headed by Nestler noted that higher dosages of D-chiro-inositol to women with PCOS without insulin resistance had minor or null efficacy than those previously investigated [42]. Also, in this study, the treatment with D-chiro-inositol obtained null or detrimental effects on hyperandrogenism. Fascinated by the research of Nestler and colleagues, Unfer was becoming interested in inositol treatments during the early 2000s. Given the huge amount of data available on D-chiro-inositol, he decided to look further on a similar compound, but with different activities. Fueled by Chiu’s findings, Unfer focused on the most abundant isomer in nature, myo-inositol. Collaborating with Baillargeon, a colleague of Nestler, Unfer tested for the first time the positive action of myo-inositol during IVF procedures.



A clinical guide to inositols

Interestingly, they noted that myo-inositol oral supplementation to women about to undergo IVF reduced the amount of exogenous FSH to be injected for stimulation [43]. This is a pivotal finding in IVF field, as myo-inositol thus reduces the risk of ovarian overstimulation, a burdensome side effect of FSH treatment. They also observed an increase in the number of retrievable oocytes, defining myo-inositol as a key molecule to achieve better outcomes from IVF procedures. Unfer and Baillargeon further collaborated to another research project. Indeed, they believed that other than D-chiro-inositol, myo-inositol could induce ovulation in anovulatory patients with PCOS as well. They treated women with PCOS with myo-inositol, expecting to induce the ovulation as in the study from Nestler and colleagues. Indeed, they achieved results comparable to those from Nestler in terms of percentage of ovulating patients, also underlining another positive effect of myo-inositol treatment in women with PCOS. In fact, Unfer pointed out an optimal recovery from hirsutism, a typical feature of hyperandrogenic women with PCOS, following myo-inositol treatment [44]. From that point on, research on inositols began gaining importance, also spanning from the classical areas of interest, namely, metabolism and gynecology. This book gathers all the most important findings obtained in recent years studying inositols and their effectiveness in different clinical settings.

References [1] Scherer J. Uber eine neue aus dem Muskelfleisch gewonnene Zuckerart. Liebigs Ann Chem 1850;73:322. [2] Maquenne L. Preparation, proprietes et constitution se l’inosite. CR Hebd Seance Acad Sci Paris 1887;104:225–7. [3] Maquenne L. Sur les proprietes de l’inosite. CR Hebd Seance Acad Sci Paris 1887;104:297–9. [4] Maquenne L. Sur quelques derives de l’inosite. CR Hebd Seance Acad Sci Paris 1887;104:1719–22. [5] Posternak S. Sur la synthese de l’ether hexaphosphorique de l’inosite avec le principe phosphoorganique de reserve des plantes vertes. C R Acad Sci 1919;169:138–40. [6] Needham J. Studies on inositol: a method of quantitative estimation. Biochem J 1923;17:422–30. [7] Posternak T. Recherches dans la serie des cyclites VI. Sur la configuration de la meso-inosite, de la scyllite et d’un inosose obtenu par voie biochimique (scyllo-ms-inosose). Helv Chim Acta 1942;25:746–52. [8] Needham J. Studies on inositol: the synthesis of inositol in the animal body. Biochem J 1924;18:891–904. [9] Folch J. Brain diphosphoninositide, a new phosphatide having inositol metadiphosphate as a constituent. J Biol Chem 1949;177:505–19. [10] Hokin LE, Hokin MR. Effects of acetylcholine on the turnover of phosphoryl units in individual phospholipids of pancreas slices and brain cortex slices. Biochim Biophys Acta 1955;18:102–10. [11] Dittmer JC, Dawson RM. The isolation of a new lipid, triphosphoinositide, and monophosphoinositide from ox brain. Biochem J 1961;81:535–40. [12] Tomlinson RV, Ballou CE. Complete characterization of the myo-inositol polyphosphates from beef brain phosphoinositide. J Biol Chem 1961;236:1902–6. [13] Berridge MJ. Discovery of the second messenger inositol trisphosphate. Messenger 2012;1:3–15. [14] Berridge MJ, Prince WT. The electrical response of isolated salivary glands during stimulation with 5-hydroxytryptamine and cyclic AMP. Philos Trans R Soc Lond Ser B Biol Sci 1971;262:111–20.

Introduction to the history of inositols

[15] Berridge MJ, Prince WT. Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J Exp Biol 1972;56:139–53. [16] Prince WT, Berridge MJ, Rasmussen H. Role of calcium and adenosine-3’:5’-cyclic monophosphate in controlling fly salivary gland secretion. Proc Natl Acad Sci U S A 1972;69:553–7. [17] Michell RH. Inositol phospholipids and cell surface receptor function. Biochim Biophys Acta 1975;415:81–147. [18] Berridge MJ, Fain JN. Inhibition of phosphatidylinositol synthesis and the inactivation of calcium entry after prolonged exposure of the blowfly salivary gland to 5-hydroxytryptamine. Biochem J 1979;178:59–69. [19] Irvine R. A tale of two inositol trisphosphates. Biochem Soc Trans 2016;44:202–11. [20] Irvine RF. A short history of inositol lipids. J Lipid Res 2016;57:1987–94. [21] Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2 + from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 1983;306:67–9. [22] Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 1984;312:315–21. [23] Cheng K, Galasko G, Huang L, Kellogg J, Larner J. Studies on the insulin mediator. II. Separation of two antagonistic biologically active materials from fraction II. Diabetes 1980;29:659–61. [24] Saltiel AR, Cuatrecasas P. Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. Proc Natl Acad Sci U S A 1986;83:5793–7. [25] Saltiel AR, Sherline P, Fox JA. Insulin-stimulated diacylglycerol production results from the hydrolysis of a novel phosphatidylinositol glycan. J Biol Chem 1987;262:1116–21. [26] Larner J, Galasko G, Cheng K, et al. Generation by insulin of a chemical mediator that controls protein phosphorylation and dephosphorylation. Science 1979;206:1408–10. [27] Larner J, Huang LC, Schwartz CF, et al. Rat liver insulin mediator which stimulates pyruvate dehydrogenase phosphate contains galactosamine and D-chiroinositol. Biochem Biophys Res Commun 1988;151:1416–26. [28] Larner J, Brautigan DL, Thorner MO. D-chiro-inositol glycans in insulin signaling and insulin resistance. Mol Med 2010;16:543–52. [29] Pak Y, Huang LC, Lilley KJ, Larner J. In vivo conversion of [3H]myoinositol to [3H]chiroinositol in rat tissues. J Biol Chem 1992;267:16904–10. [30] Asplin I, Galasko G, Larner J. chiro-inositol deficiency and insulin resistance: a comparison of the chiroinositol- and the myo-inositol-containing insulin mediators isolated from urine, hemodialysate, and muscle of control and type II diabetic subjects. Proc Natl Acad Sci U S A 1993;90:5924–8. [31] Ortmeyer HK, Huang LC, Zhang L, Hansen BC, Larner J. Chiroinositol deficiency and insulin resistance. II. Acute effects of D-chiroinositol administration in streptozotocin-diabetic rats, normal rats given a glucose load, and spontaneously insulin-resistant rhesus monkeys. Endocrinology 1993;132:646–51. [32] Nestler JE. Stimulation of androgen production by insulin-like growth factor I. Ann Intern Med 1986;105:796. [33] Nestler JE, Clore JN, Strauss 3rd JF, Blackard WG. The effects of hyperinsulinemia on serum testosterone, progesterone, dehydroepiandrosterone sulfate, and cortisol levels in normal women and in a woman with hyperandrogenism, insulin resistance, and acanthosis nigricans. J Clin Endocrinol Metab 1987;64:180–4. [34] Nestler JE. Modulation of aromatase and P450 cholesterol side-chain cleavage enzyme activities of human placental cytotrophoblasts by insulin and insulin-like growth factor I. Endocrinology 1987;121:1845–52. [35] Nestler JE, Romero G, Huang LC, Zhang CG, Larner J. Insulin mediators are the signal transduction system responsible for insulin’s actions on human placental steroidogenesis. Endocrinology 1991;129:2951–6. [36] Nestler JE, Jakubowicz DJ, de Vargas AF, Brik C, Quintero N, Medina F. Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system. J Clin Endocrinol Metab 1998;83:2001–5.



A clinical guide to inositols

[37] Chiu TT, Rogers MS, Law EL, Briton-Jones CM, Cheung LP, Haines CJ. Follicular fluid and serum concentrations of myo-inositol in patients undergoing IVF: relationship with oocyte quality. Hum Reprod 2002;17:1591–6. [38] Felch WC, Dotti LB. Depressing effect of inositol on serum cholesterol and lipid phosphorus in diabetics. Proc Soc Exp Biol Med 1949;72:376–8. [39] Felch WC, Keating JH, Dotti LB. The depressing effect of inositol on serum cholesterol and lipid phosphorus in hypercholesteremic myocardial infarct survivors. Am Heart J 1952;44:390–5. [40] Nestler JE, Jakubowicz DJ, Reamer P, Gunn RD, Allan G. Ovulatory and metabolic effects of D-chiro-inositol in the polycystic ovary syndrome. N Engl J Med 1999;340:1314–20. [41] Iuorno MJ, Jakubowicz DJ, Baillargeon JP, et al. Effects of d-chiro-inositol in lean women with the polycystic ovary syndrome. Endocr Pract 2002;8:417–23. [42] Cheang KI, Baillargeon JP, Essah PA, et al. Insulin-stimulated release of D-chiro-inositol-containing inositolphosphoglycan mediator correlates with insulin sensitivity in women with polycystic ovary syndrome. Metabolism 2008;57:1390–7. [43] Papaleo E, Unfer V, Baillargeon JP, Fusi F, Occhi F, De Santis L. Myo-inositol may improve oocyte quality in intracytoplasmic sperm injection cycles. A prospective, controlled, randomized trial. Fertil Steril 2009;91:1750–4. [44] Papaleo E, Unfer V, Baillargeon JP, et al. Myo-inositol in patients with polycystic ovary syndrome: a novel method for ovulation induction. Gynecol Endocrinol 2007;23:700–3.


Physiological and pathophysiological roles of inositols Giovanni Monastraa,b, Simona Dinicolaa,b, and Vittorio Unfera,b a Systems Biology Group Lab, Rome, Italy The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy


Introduction Inositols (Ins), belonging to the group of sugar alcohols (cyclic polyols or hexahydroxycyclohexanes), are white crystals, odorless, and with a slightly sweet taste, soluble in water and insoluble in absolute alcohol and ether. Of note, they are stable to heat, strong acids, and alkalis. In this group of compounds, myo-inositol (myo-Ins), which has deep structural similarities to glucose, is the most important and widespread, and it is detectable ubiquitously in almost all biological systems. Inositol molecule is an essential growth factor for many cells in tissue culture. Its derivatives, in particular phosphates, also perform crucial physiological functions [1,2]. The first scientific investigations on inositol date back to the mid-nineteenth century, when the German physician and chemist Johann Joseph Scherer published a paper describing a hexahydroxycyclohexane obtained from muscle cells, called “inositol.” This name is derived from the combination of some Greek words [in-, “sinew, fiber,” -ose (indicating a carbohydrate), -ite (“ester”), -ol (“an alcohol”)] [3]. Only many years later, the French chemist and plant physiologist LeonGervais-Marie Maquenne defined the inositol structure purifying it from leaves and, in the subsequent experiments, using large quantities of horse urine reduced by boiling [4–6]. Then, the Swiss chemist Theodore Posternak, famous for his long and in-depth research on inositols, was able to elucidate the three-dimensional structure of myo-Ins. We owe him the discovery of inositol phosphates about a century ago. He also identified phytic acid as the major store of organic phosphate in seeds [7,8]. Here, we provide in advance some brief general information on inositol activity useful to the reader as landmarks in such a complex topic. Almost all inositols exert an insulin-mimetic activity and an insulin-sensitizing effect, although with different effectiveness. Also, they have a huge number of other important physiological activities, such as promoting ovulation and fertility. Several inositols cross the cell membrane by two cation-coupled cotransporters (sodium or proton coupled) and become components of cellular metabolism. It is important to keep in mind that inositols can also act through a receptor pathway. This occurs only when these molecules are joined to one or more A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00008-X

Copyright © 2023 Elsevier Inc. All rights reserved.



A clinical guide to inositols

phosphoric groups, forming inositol phosphates, which have a specific intracellular receptor to transmit the signal [9–17].

Evolution of inositols From the evolutionary point of view, myo-Ins is a very ancient molecule and its biosynthesis was recognized as a well-conserved pathway. myo-Ins appeared very early and can be defined as prebiotic “Ur” compound [18] since it was already present in the prebiotic era. Unlike acyclic polyols and reducing sugars, myo-Ins shows per se a great stability. Indeed, its chemical features preserve it from degradation caused by high temperatures or oxidation. Clearly, such a condition was necessary to face and withstand the extreme environmental challenges that occurred before the appearance of life. Some persuasive speculations [19] connect the evolution of the physiological functions performed by myo-Ins with those of phosphorus, which, in form of phosphate, plays a key function in living beings. Therefore, phosphate could have been incorporated early into the primitive molecules. As Saiardi suggested, we should look retrospectively to phosphate-rich molecules present in cells today. The current sophisticated association of inositol with phosphate, and the roles of some inositol phosphates in regulating cellular phosphate homeostasis, suggest that myo-Ins and its derivatives might have played some role in the prebiotic process of phosphate exploitation. Due to myo-Ins stability, this molecule represents the best candidate for the initial organophosphate molecules, such as primitive inositol phosphates. The possible prebiotic synthesis of inositol pyrophosphates could have given rise to high-energy chemical compounds, already useful in the first transphosphorylating processes [19]. However, we are still on the level of hypotheses, albeit very stimulating. It was discovered that many Archaea produce and use inositols [20], also to get the thermoprotective solute di-L-Ins-1,10 -phosphate, a molecule playing the role of osmolyte in hyperthermophilic Archaea [21]. According to Michell [1], it seems likely that early Archaea developed myo-Ins synthesis from glucose-6-phosphate and used it as membrane phospholipid headgroup at the beginning of their evolution [1]. All organisms producing inositol utilize evolutionarily related myo-inositol monophosphate synthases (often abbreviated as Ins3P synthases or MIPS) [2,22–27]. Most of the archaeal genomes encode the enzyme Ins3P synthase, and many archaeons incorporate the resulting inositol into their characteristic sn-1-phosphoryl-2,3-diether-based membrane glycerolipids [28]. The next step in evolution occurred when the ability to synthesize myo-Ins was incorporated by early eukaryotes by means of horizontal gene transfer. Soon enough, eukaryotes established ubiquitous functions for phosphoinositides in membrane trafficking and Ins polyphosphate synthesis. Finally, approximately 1000 million years ago, Ins(1,4,5)P3 receptor Ca++ channels and the role of messenger of Ins(3,4,5)P3 emerged by functional diversification in amoebozoans and metazoans [1]. On the contrary, a different landscape is found among bacteria. In fact, mainly, they exploit inositols as a carbon

Physiological and pathophysiological roles of inositols

source; nevertheless, few of them synthesize these compounds or use them. In this small group of bacteria, there are hyperthermophilic Thermotogales, which use di-L-1,10 phosphate to survive in extremely hostile environments. In fact, the upper-temperature border of growth in the genus Thermotoga is 90°C. These bacteria, together with the members of Aquificales, show the highest growth temperatures known so far. Another group able to use myo-Ins is the actinomycetes such as Mycobacterium spp., which take advantage from mycothiol, an inositol-containing thiol, as an intracellular redox reagent. Bacteria also acquired their Ins3P synthases by horizontal gene transfer from Archaea. It is worthy of note that stressed plants, insects, deep-sea animals, and kidney tubule cells are able to face and overcome environmental variation by making or accumulating different inositol derivatives. Eukaryotes utilize phosphatidylinositol (PI) derivatives for cell signaling and regulation and for protein anchoring at the cell surface. Of note, the diradylglycerol cores of archaeal and eukaryote/bacterial glycerophospholipids have mirror image configurations: sn-2,3 and sn-1,2, respectively [1]. Finally, it seems that phosphatidylinositol 3-phosphate (PI3P) was the first polyphosphoinositol lipid to emerge during evolution [29].

Activity of myo-Ins and its stereoisomers In its free form, myo-Ins acts as an osmolyte involved in cytoprotection, and this was its first physiological function emerging during evolution. In this field, the effects of this molecule include osmotic compensation, protein stabilization, and freezing avoidance when tissue water is strongly cooled [30]. D-Chiro-inositol (D-chiro-Ins), a stereoisomer of myo-Ins, is another inositol of great interest. The endogenous production of these two stereoisomers fluctuates depending on the specific tissue needs [31]. For example, in healthy women, the plasma ratio is 40:1 [32], whereas in ovarian follicular fluid, it is close to 100:1 [33]. Overall, there are seven myo-Ins stereoisomers that are naturally formed (scyllo-, muco, epi-, neo-, allo-, D- and L-chiro-inositols), by means of different isomerases, and one, cis-inositol, whose existence in nature is unknown [34]. Between 7% and 9% of myo-Ins is transformed into D-chiro-Ins as demonstrated using the radiolabeled [3H]-compound; instead, the other stereoisomers resulted very low, not more than 0.06% of total radiolabeled myo-Ins [35]. Some body districts, such as brain, heart, and ovary, utilize high quantity of glucose; for this reason, they show significant amounts of myo-Ins in respect to other tissues [36]. For example, the brain contains levels of myoIns 10- to 15-fold higher than those in the blood [37]. In turn, D-chiro-Ins is obtained from myo-Ins by a unidirectional reaction. The key enzyme deputy to catalyze this process is a tissue-specific nicotinamide adenine dinucleotide (NAD)-NADH-dependent epimerase, which works under stimulation by insulin [38]. In this way, each organ



A clinical guide to inositols

and tissue takes advantage from a specific and proper ratio between myo-Ins and D-chiro-Ins content, necessary to maintain the physiological and healthy conditions [39].

Glycemic control One of the most significant activities exerted by myo-Ins and D-chiro-Ins concerns glycemic regulation. Both stereoisomers exert an insulin-mimetic activity and are also effective against insulin resistance [31,40]. Glycan-containing D-chiro-Ins (IPG-P) shifts glucose metabolism toward glycogen synthesis, while glycan-containing myo-Ins (IPG-A) shifts it toward glucose catabolism. However, it is very difficult to drastically separate their individual actions as the two stereoisomers are metabolically linked together. myo-Ins, as inositol 1,4,5-trisphosphate (InsP3), increases pyruvate dehydrogenase (PDH) activity, which induces glycolysis and Kreb’s cycle, resulting in the production of adenosine triphosphate (ATP), thus improving glucose utilization [41]. myo-Ins seems to exert its effects at different levels of glucose concentrations in the body. It significantly inhibits the duodenal glucose absorption and reduces glucose rise in blood. This finding can be explained by an interference of myo-Ins on glucose intestinal absorption [42]. Moreover, myo-Ins improves insulin sensitivity in adipocytes by increasing lipid storage capacity and glucose uptake, and by preventing lipolysis [43]. myo-Ins acts also through the expression of peroxisome proliferator-activated receptor γ (PPAR-γ), a type II nuclear receptor (protein-regulating genes) controlling a large number of metabolic pathways where myo-Ins is involved. PPAR-γ, along with α and β forms, is considered the master regulator of adipogenesis. In humans, PPAR-γ is most highly expressed in the adipose tissue and less in the skeletal muscle, colon, and lungs [44]. Target genes of PPAR-γ are involved in glucose metabolism [45], adipocyte differentiation, and lipid storage [46]. In liver, PPAR-γ contributes to triglyceride homeostasis and protects other tissues from insulin resistance [47] and triglyceride accumulation [48]. Furthermore, it downregulates the inflammatory response, mainly in macrophages, likely through the inhibition of proinflammatory transcription factors (e.g., STAT, NF-κB, and AP-1) [49]. In addition, myo-Ins induces the translocation to the plasma membrane of GLUT4 transporters, which are expressed in intracellular vesicles, leading to the increase in glucose uptake and the decrease in plasma glucose level under glucose-loaded hyperglycemic condition [50,51]. This effect mimics the insulin action, since insulin induces GLUT4 translocation from the endoplasmic reticulum to the plasma membrane to stimulate glucose uptake in skeletal muscle cells [52]. As expected, the impairment of this activity elicits insulin resistance [53]. myo-Ins can also directly promote the activation of insulin receptor substrate (IRS) and Akt [54]. Furthermore, myo-Ins decreases the free fatty acid release from adipose tissues by the inhibition of adenylate cyclase [39,55]. Also, D-chiro-Ins causes GLUT4 translocation to the plasma membrane [56] and is involved in stimulating PDH [57,58]. Moreover, D-chiro-Ins increases mRNA and protein

Physiological and pathophysiological roles of inositols

expression of IRS2, PI3K, and Act, upregulating P-Act protein and downregulating GSK3β levels [59]. All of them are essential players in the signal transduction of insulin and other hormones. Josef Larner and colleagues were the first to suggest that inositols play the role of insulin mediators [58,60,61]. This group of researchers isolated and purified two glycans containing D-chiro-Ins and myo-Ins. When administered in vivo, these molecules demonstrated to have insulin-mimetic properties. Indeed, when injected intravenously, they dose dependently decreased hyperglycemia in streptozotocininduced diabetic rats, whereas intraperitoneally administered in rats, they stimulated labeled glucose incorporation into glycogen in the diaphragm muscle [62]. In independent experiments, also John Nestler with his collaborators confirmed the insulin-mimetic activity of a glycan-containing D-chiro-Ins (called INS-2) on human ovarian thecal cells [63,64]. They observed that both insulin and D-chiro-Ins induce testosterone biosynthesis, effect that was prevented by an antibody directed against this glycan. Other groups showed that inositol-derived phospholipids (see further on), such as InsP3 [65], InsP2 [66], and InsP7 [67], act to allosterically control insulin signaling.

Human reproduction Besides glycemic control, inositols play an essential function in the physiology of female and male reproduction. In women, myo-Ins (as InsP3) is one of the second messengers of FSH, and therefore, it is involved in regulating proliferation and maturation of granulosa cells. Due to this role, myo-Ins modulates the production of anti-Mullerian hormone (AMH), and consequently determines oocyte maturation and transport in the oviduct as well as guarantees the formation of good-quality embryos [68]. As mentioned before, ovaries are characterized by the specific ratio not too much lower than 100:1 between myo-Ins to D-chiro-Ins, which ensures their best physiological condition. Ravanos and colleagues demonstrated that myo-Ins levels in the reproductive tract of healthy females should be substantially higher than D-chiro-Ins, supporting its important role in the ovaries. Under normal homeostatic conditions, ovarian myo-Ins/D-chiro-Ins ratio ranges from 70:1 to 100:1. In fact, higher D-chiro-Ins concentrations negatively impact on oocyte and blastocyst quality [69]. Hence, any D-chiro-Ins excess must be absolutely avoided, also in consideration of the influence exerted by this stereoisomer on steroidogenesis, a research area that should deserve much more consideration than it had so far. Indeed, both myo-Ins and D-chiro-Ins have a great impact on the androgenic and estrogenic pools with serious negative consequences in case of an imbalance between them. We know that D-chiro-Ins stimulates the ovarian production of androgens by thecal cells. In 1998, Nestler first demonstrated that D-chiro-Ins increases testosterone concentrations in theca cells from PCOS women [70]. Recently, a new study indicated that D-chiro-Ins directly regulates the gene expression of enzymes involved in steroidogenesis in human granulosa cells, dose dependently reducing the expression of both aromatase and



A clinical guide to inositols

cytochrome P450 side-chain cleavage genes [71]. Through this newly discovered mechanism, D-chiro-Ins reduces estrogen levels without completely preventing their biosynthesis. On these premises, it was speculated that despite their chemical similarities, myo-Ins to D-chiro-Ins in many cases play different physiological roles. Concerning their effects on aromatase activity, it was speculated that myo-Ins acts in an opposite manner with respect to D-chiro-Ins. Higher myo-Ins/D-chiro-Ins ratios could increase the activity of aromatase in granulosa, inducing estrogen biosynthesis, whereas we know that the lowering of this ratio stimulates androgen production in thecal cells. In fact, as mentioned before, myo-Ins (in the form of InsP3) is a second messenger of FSH, which is reported to be the main stimulator of aromatase activity in granulosa cells [72,73]. Furthermore, myo-Ins could modulate ovarian steroidogenesis by rearranging cytoskeletal proteins [74]. In conclusion, the increased D-chiro-Ins concentration promotes androgen synthesis, whereas myo-Ins reduction worsens the energy state of the oocytes, impairing FSH signaling and oocyte quality [69].

myo-Ins-derived phospholipids Eukaryotic cells employ myo-Ins backbone to generate different types of signaling molecules, which play key roles in essential physiological processes, namely, when the cell faces external stressors [75]. To do this, the cells assemble phosphate groups around the six-carbon inositol ring. myo-Ins-derived phospholipids or inositol phosphates (InsPs), such as InsP2, InsP3, InsP4, InsP5, and InsP6, are produced by phosphorylation of one or several of its hydroxyl groups. Most of these molecules are not present in mammal cells; instead, they can be found in plants. myo-Ins is absolutely the main molecule used to synthesize phospholipids; however, some phosphatidyl-scyllo-Ins or phosphatidyl-chiro-Ins can sporadically be detected in plant and animal cells [1,76]. All biological processes take advantage of the exceptionally varied structural organization of phosphorylated inositols. Phosphates can form three distinct covalent bonds with inositol: phosphoester, phosphodiester, and phosphoanhydride bonds, with each providing different chemical features to the molecule [77]. The numerous derived forms include the above-mentioned InsPs, phosphatidylinositol, phosphatidylinositides (phosphatidylinositol-phosphates [PIPs]), glycosyl-phosphatidylinositols (GPI), and many other derivatives as inositol phosphoglycans (IPG) and Ins ethers and esters [78]. The study of their physiological activities is complicated by some factors [75]: they are a multitude (inositol monophosphates alone have 63 possible isomers) and participate, also as second messengers, in a multiplicity of metabolic and signaling pathways (particularly due to their involvement in Ca2++ metabolism). All in all, InsPs convey signals for a variety of growth factors, hormones, and neurotransmitters [79]. In any case, we can schematize by saying that myo-Ins and its derivatives, particularly its phosphates InsP3 and InsP6, exert physiological roles in various taxa [1,80,81], including the regulation of ion channel

Physiological and pathophysiological roles of inositols

permeability [12,16,82–84], phosphate levels [85], metabolic flux [86,87], transcription control, mRNA export and translation, DNA repair [10,88], insulin signaling, embryonic development [10], and stress response [89]. myo-Ins is also a component of membrane-incorporated phosphatidyl inositols [90]. It is noteworthy that these small signaling molecules take part in the tight regulation of a huge number of cellular activities. Humans can produce InsPs by the dephosphorylation of more phosphorylated molecules, such as InsP6, by specific phosphatases, and/or by phosphoinositides hydrolysis [91,92]. Plants and protozoa obtain them through direct phosphorylation of myo-Ins by a specific kinase [93,94] that is lacking in human cells. In fact, after stimulation, phospholipase C metabolizes PI (4,5)P2 into two molecules: the intracellular second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3 or IP3). DAG is an activator of protein kinase C (PKC), whereas InsP3 plays an essential role in calcium release from endoplasmic reticulum (ER) to the cytosol and mitochondria, thus regulating metabolism and cell fate [91,92,95,96]. Such release is stimulated by the binding of Ins(1,4,5)P3 ligands to its receptor, constituted by type I, II, and III distinct isoforms [82,97]. InsP3 regulates cell proliferation and other cellular reactions that require free calcium, for example, the contraction of the muscle cells. After intracellular Ca2+ depletion, the replenishment of its reserves is achieved through a process called capacitive Ca2+ entry. Ins(1,4,5)P3 has a short half-life within the cell [98] and is rapidly metabolized to free myo-Ins and inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4 or InsP4) [99]. Moreover, other InsPs and diphosphoryl InsPs (called inositol pyrophosphates or InsP7 and InsP8) were also discovered in the cells [100]. Up to date, we can state that InsPs regulate each aspect of cellular physiology [77]. The recycling of InsPs by a series of InsPphosphomonoesterases to regenerate the intracellular pool of free myo-Ins is a critical pathway, as it represents the main source of myo-Ins during endocrine stimulation. Cells actively preserve InsP4 concentrations, given that animals feeding an InsP6-free diet have unchanged InsP4 levels, while InsP6 and InsP5 are markedly reduced [101]. Furthermore, similar results were obtained in mold, where myo-Ins starvation does not significantly change InsP4 levels [102]. InsP4 consists of a group of several different isomers: 3,4,5,6-InsP4, 1,4,5,6-InsP4, 1,3,4,5-InsP4, and 1,3,4,6-InsP4. The first three of them regulate chloride ion channels, histone acetylation, and calcium signaling, respectively [10,103]. Furthermore, myo-Ins and D-chiro-Ins contribute to the formation of glycosyl-phosphatidylinositols (GPIs) anchors and of inositol phosphoglycans (IPGs) that are putative second messengers of insulin action in the GPI/IPG pathway [55]. Stimulation by insulin causes the hydrolysis of GPI lipids situated on the outer leaflet of the cell membrane. This allows the release of IPG-containing myo-Ins or D-chiro-Ins. Noteworthy, IGPs is deeply involved in intracellular metabolism, in particular through the activation of key enzymes that control the oxidative and non-oxidative metabolism of glucose. IPGs containing D-chiro-Ins or myo-Ins significantly improve glucose metabolism, decreasing insulin resistance (IR) [104]. Some studies demonstrated that non-phosphoglycans—as



A clinical guide to inositols

pyrophosphate diphosphoinositol pentakisphosphate, or InsP7, and phosphatidylinositol 5-phosphate, or PI5P—are also capable to modulate glucose metabolism. InsP7 is necessary for the exocytosis of insulin-containing secretory granules from pancreatic β cells [105], whereas PI5P mimics insulin effect in enabling GLUT4 translocation to cell surface and improving glucose uptake [106].

Natural sources of inositols Adequate amounts of myo-Ins can be introduced into the body through the diet. It is reported that human adults consume approximately 1 g of this compound per day in different biochemical compositions [78]. myo-Ins is found in the human diet in its free form or as inositol-containing phospholipid in animal-derived foods, or as phytic acid (inositol-hexakisphosphate or InsP6) or phytates in plant foodstuffs. Cereals and legumes contain high concentrations (0.4%–6.4%) of InsP6, which is the major form of phosphorous storage in seeds. Another form can be found in deposits of mixed “phytate” salts of K+, Mg2+, Ca2+, Mn2+, and Zn2+ [1,10]. In the plant kingdom, it is also found in other plant tissues and organs such as pollen, roots, tubers, and turions. InsP6 accumulates during seed development and is broken down into lower inositol phosphates during germination. The cytosol of almost all mammalian cells contains InsP6 and its lower phosphorylated forms (InsP1–5) as well as myo-Ins. The major source of myo-Ins is likely to be phytic acid, one of the most interesting bioactive food compounds for its anticancer, antioxidative, and anticalcification effects. myo-Ins derivatives constitute the main storage manner of phosphorus in several vegetable tissues. And here we recall the hypothesis of an evolutionary link between myo-Ins and phosphorus. Bacterial phytases and phosphatases are principally responsible for the digestion of dietary InsP6, followed by the release of myo-Ins and phosphate [107,108]. Dietary needs may be highly depending on the consumer’s age, prolonged antibiotic use, or the frequent daily drinking of coffee (more than 100 mg per day, that are approximately equivalent to two espresso cups) [109]. In addition, we should consider that since the 1970s phytic acid was removed from a lot of foods. In fact, under conditions of poor diet, this substance, due to its molecular structure, is an antinutrient, which gives rise to insoluble complexes with essential divalent cations (chelating capacity), such as iron, calcium, and zinc, reducing their bioavailability [110]. However, the antinutrient effect of phytic acid only occurs when significant quantities of this substance are consumed with a diet having very small amount of the essential trace elements for the human diet. In fact, unbalanced nutrition or undernourishment can induce serious deficiencies, a condition of particularly great significance for developing countries. However, this is not the case of Western nations, where “antinutrient” referred to phytic acid is now an outdated term. In industrialized countries, the beneficial properties of phytic acid are very important, also in consideration of the huge problems due to civilization diseases. Therefore, we can speculate, although

Physiological and pathophysiological roles of inositols

controversial, that in the industrialized countries low-vegetable consumers may suffer from a relative deficiency of phytic acid and derivatives thereof, caused by its reduced content in the diet [111,112]. There are few studies on the dietary sources of inositols. The first one was carried out by Clements and Dornell [113], who observed that myo-Ins content significantly differs among the food assortments as well as within each one. Milk showed a relatively low total myo-Ins content. Among the vegetables, the highest contents were found in the beans and the lowest contents in the leafy vegetables. In general, fresh vegetables contain higher myo-Ins levels than frozen, canned, or salt-free products. Extremely high contents of myo-Ins can be detected in cantaloupe and the citrus fruits (except for lemons). Whole grain breads have more myo-Ins than refined breads, while among the cereals, oats and bran contained more of this molecule than cereals derived from other grains. The authors also found considerable variation of myo-Ins content in starchy vegetables. Meat and fat show relatively low myo-Ins levels. Thus, it appears that food consisting in seeds (beans, grains, and nuts) provides the most concentrated source of dietary myo-Ins, while other foods contain rather modest amounts of it. We wish to point out that, in the study by Clements and Darnell [113], most participants defined the high-myo-inositol diets as highly palatable. Another study [114] offers some new data, concerning the myo-Ins content in the form of phytic acid, namely, myo-Ins hexaphosphate. As example, phytic acid content (on the dry matter) is 0.2%–9.4% in nuts, with higher levels in almonds (9.42%) and walnuts (6.69%), 0.4%–5.7% in oil seeds, 0.2%–2.4% in legumes, and 0.04%–3.3% in cereals. For a healthy diet, we should keep in mind that in cereals, most of the phytates are in the aleurone layer, the outermost sheet of the endosperm, followed by the inner starchy endosperm. Instead, the endosperm is almost devoid of phytates [108], and this is the reason why whole-grain cereals contain much more phytates than processed cereals.

Inositol endogenous synthesis and catabolism The first studies on inositol endogenous synthesis carried out in the 1960s showed that rat testis, brain, kidney, and liver synthesize inositol from glucose [115–117]. The research by Clements and Diethelm [118] tried to evaluate the in vivo rate of inositol production in the human kidney. It was estimated that one normal kidney can endogenously synthesize around 2 g per day. It means that the body in a binephric human produces at least 4 g of new inositol per day, which largely exceeds its daily amount introduced with diet (1 g). Other tissues also can give a contribution to the endogenous production of inositol in humans and animals. In fact, the rabbit brain synthesizes in situ from glucose approximately one half of its unbound inositol, whereas the rest comes from the blood [119]. All biological myo-Ins is made by a single type of enzyme, the previously mentioned Ins3P synthase [27]. The main cytosolic enzyme [120] is NADH-dependent cycloaldolase, which converts the ubiquitous central metabolite glucose-6-phosphate,



A clinical guide to inositols

via a 5-ketoglucose-6-phosphate intermediate, to Ins3P (which is synonymous with L-myoinositol-1-phosphate and D-myo-inositol 3-phosphate) [1]. Once produced, the Ins3P is then dephosphorylated by the enzyme inositol monophosphatase (IMPA-1 or IMPase) to give free myo-Ins [28]. In addition, other pathways emerged to get free inositol, taking advantage of the dephosphorylation of InsP3 and InsP2 [121]. myo-Ins catabolism starts with the cleavage of the ring to obtain O-glucuronic acid and, after other metabolic steps, oxylulose-5-phosphate which enters the pentose phosphate cycle. myoIns catabolism occurs mainly in the kidney; indeed in nephrectomized animals, its degradation is impaired with considerable anomalies in myo-Ins metabolism and high plasma concentrations of this molecule [122].

Inositol transport system The absorption of inositol can occur by a passive diffusion process when this molecule is highly concentrated. However, the main mechanism is a very complex system of transporters found in bacteria, protozoa, fungi, plants, and animals. Such system of transporters plays a crucial role in the intestinal absorption and selective cell uptake from blood and allows to obtain an unequal intracellular distribution of myo-Ins, D-chiro-Ins, and similar molecules. To do this, inositol passage and storage occur against a concentration gradient by means of active transport, discovered for the first time by Caspary and collaborators [123]. This process is unaltered by high dietary calcium intake [124] but is significantly reduced by glucose and other sugars [125–128]. Starting from the early 1990s, as described in the masterful review by Schneider [129], new studies allowed to better understand how inositol transporters function. Researchers found out that there are two types of transporters: Na+-coupled, and H+-coupled. The first identified Na+-coupled transporter was responsible for the accumulation of myo-Ins in canine kidney cells exposed to hypertonic medium. The protein showed high similarity with Na+/glucose transporters [130]. Later, the same kind of transporter was also detected in rat [131], human [132], and mouse [133], and it was named SMIT1 after the discovery of a second Na+-coupled inositol transporter [134], known as SMIT2. Both protein transporters are located in the plasma membrane [135], and the genes encoding Na+-coupled inositol transporters have similar expression patterns. The gene encoding SMIT1 was detected in kidney, brain, placenta, pancreas, heart, skeletal muscle, lung, and bones [132,136,137]. On the other hand, the gene encoding SMIT2 is highly expressed in kidney, heart, skeletal muscle, liver, placenta, spleen, leukocytes, and weakly expressed in the brain [138,139]. Furthermore, it was found in small intestine [140] and oocytes [141]. myo-Ins is the preferred substrate of SMIT1 with Km ¼55 μM [142]. Coady and colleagues demonstrated that SMIT2 accepts D-chiro-Ins as well as myo-Ins, with Km values of 130 and 120 μM, respectively [138]. The same authors hypothesized that myo-Ins is the physiological substrate for SMIT2 since D-chiro-Ins concentration in

Physiological and pathophysiological roles of inositols

plasma is rather low [138]. Both transporters show only a low affinity for glucose (Km 50 mM for SMIT1 and 30 mM for SMIT2) [138,140,142,143]. D-Chiro-Ins can compete with myo-Ins for SMIT2-mediated transport. Of note, in cultured human fibroblasts as well as in MDCK cells isolated from normal kidney tissue from an adult, female cocker spaniel, the downregulation of protein kinase C activity increases myoIns transport, while the activation of protein kinase A decreases it. This finding led to consider that inositol uptake is regulated at posttranslational level by phosphorylation [144,145]. The second type of inositol transporter, named HMIT, was identified as a proton-coupled protein in 2001. It is a H+/myo-inositol symporter with a Km of 100 μM. Also scyllo-, chiro-, and muco-inositols, but not glucose or fructose, utilize HMIT as transporters [146]. HMIT is predominantly expressed in brain, and at lesser extent in adipose tissue kidney [146] and oocytes [141]. Concerning myo-Ins uptake by oocytes, Pesty et al. for the first time have demonstrated this active process in maturing mouse oocytes since 1994 [147]. As highlighted above, this active transport system is very complex and articulated. It allows the compartmentalization and accumulation of inositols in specific areas of the body, a condition that is essential for maintaining the correct physiological balance of the organism.

Inositols levels in plasma and tissues The normal circulating fasting plasma myo-Ins concentration in adults is between 13 and 43 μM [113,148–152]. In utero, the early fetal plasma myo-Ins levels are 2–10 times higher and gradually decrease toward term [153]. There is no appreciable difference between myo-Ins values detected in whole blood and in plasma [154,155]. Instead, the values for D-chiro-Ins ranged between 0.15 and 0.90 μM in men [152] and between 0.33 and 9.8 in women [151]. Furthermore, since the early 1960s, it is well known that some organs selectively store myo-Ins [155]. Obviously, the selective storage found in different tissues is strongly in favor of the active myo-Ins transport against gradient. In experimental animals (rat, guinea pig, and rabbit), free myo-Ins level in all the tissues is appreciably above that in the plasma. Noteworthy, even if with significant variations among the three species examined, in kidney cortex, testes, ovary, thyroid, adenohypophysis, spleen myo-Ins is about 10–100 times higher in respect to the respective plasma values [154,156,157]. Male reproductive tract, together with epididymal, vesicular, and prostatic fluids, is rich in free myo-Ins [158]. Elevated myo-Ins concentrations, several fold greater than in blood, have also been confirmed in mammalian semen [159]. Unbound myo-Ins levels in the brain, cerebrospinal fluid, and choroid plexus are also higher than in plasma [119]. While in the liver, muscle, and heart, myo-Ins is predominantly represented in its phospholipid-bound form [160], high free myo-Ins levels were recorded in the kidney, small intestine, and brain. During lactation, breast milk contains a



A clinical guide to inositols

rich supply of myo-Ins [161–163] and a deprivation of this molecule in pups causes a deficiency in several tissues, with an important exception in the nervous system. In fact, as consequence of myo-Ins deprivation from the diet, plasma, testis, lens, liver, heart, lung, kidney, and small intestine showed a significant decrease in free myo-Ins levels, in comparison with those recorded in normally supplemented pups. Instead, myo-Ins amount in the neural tissues of these animals was unaffected by its nutritional deficiency [164]. This evidence shows that the nervous tissue is the only area of the body which absolutely needs myo-Ins and that it has an endogenous system of synthesis able to guarantee its proper levels.

Inositols: From the physiology to the pathophysiology As previously mentioned, the physiological levels of the two main inositols, namely, myo- and D-chiro-, are closely related to the conversion process operated by a specific enzyme. Indeed, under insulin stimulation, a tissue-specific epimerase converts myo-Ins into D-chiro-Ins according to tissue requirements [38]. This unidirectional reaction allows each organ and tissue to benefit from a specific and proper balance between these two stereoisomers, ensuring the correct metabolic functions and consequent physiological status [57]. Pathologic conditions of insulin resistance decrease insulin sensitivity in many tissues leading to a reduced epimerase activity and, consequently, lowering D-chiro-Ins production [38,40,165]. Paradoxically, ovaries, differently from most tissues, can maintain their normal insulin sensitivity. For this reason, the systemic compensatory hyperinsulinemia overstimulates epimerase activity in those tissues, causing excessive D-chiro-Ins synthesis at the expense of myo-Ins concentration [166]. Therefore, insulin resistance is responsible for, or alternatively contributes to, the unbalance of myo-Ins-to-D-chiro-Ins ratio and may account for those pathologic conditions associated with this kind of metabolic disturbance, especially PCOS [167]. Indeed, the altered ovarian myo-Ins/D-chiro-Ins ratio can explain the pathogenesis of PCOS in insulinresistant patients. Evidence supporting such theory was provided by two studies. In the first one, the authors analyzed the epimerase activity and the content of myo-Ins and D-chiro-Ins in PCOS theca cells [57], while in the second, Unfer et al. investigated the concentration of the two inositols in the follicular fluid (FF) of healthy and PCOS women [33]. Both studies obtained similar results. Namely, the ovaries of healthy women presented higher concentrations of myo-Ins and lower concentrations of D-chiro-Ins; however, the ovary of PCOS patients showed the opposite situation, with a marked depletion of myo-Ins and an increased D-chiro-Ins content. This increase in D-chiro-Ins concentration induces androgen synthesis and accumulation [64,71]; meanwhile, myo-Ins depletion worsens FSH signaling and oocyte quality. Overall, these are the main phenotypical features of PCOS clinical picture.

Physiological and pathophysiological roles of inositols

Chiu was the first to observe a positive correlation between increased concentrations of myo-Ins in FF and better quality of the oocytes [168]. That finding prompted to state that higher levels of myo-Ins in FF may be related to the well-being of follicle and quality of oocytes and embryos [168]. The same author suggested that meiotic maturation and subsequent potential development of the oocyte may be increased by myo-Ins availability [169]. Thus, myo-Ins can predict the high quality for oocyte evaluation, while the increase of D-chiro-Ins concentrations definitely worse the oocyte quality. The study by Ravanos et al. confirmed the importance of the different roles exerted by myo-Ins and D-chiro-Ins and found out a correlation between myo-Ins/D-chiro-Ins ratio in FF and blastocyst quality. The value of this ratio, ranging from 100:1 to 70:1 in favor of myo-Ins, may represent a new biomarker for estimating the good features of blastocysts, and also a prognostic factor for embryo implantation and pregnancy success [69]. Besides metabolic alterations, myo-Ins depletion may also expose patients to several neuropathological and psychiatric conditions, including Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclerosis and depression, suggesting a protective role for myoIns in different neurodegenerative and neurological disorders [170]. Furthermore, a depletion of myo-Ins may also negatively influence fetus physiological development, predisposing the newborn to unfavorable outcomes, like neural tube defects (NTDs) [171]. Indeed, studies in rodent embryos have shown that inositol deficiency resulted in cranial NTDs in cultured embryos [172–174], indicating a crucial role for inositol in neural tube closure. Also in humans, lower serum myo-Ins concentrations have been reported in mothers of children with spina bifida [175], suggesting a possible predisposing association. Moreover, inositol promotes the maturation of several components of pulmonary surfactant and may play a critical role in fetal and early neonatal life. A drop in myo-Ins levels in infants with respiratory distress syndrome (RDS) can be a sign that their illness will be severe [176]. Notably, for its central role as a second messenger in the thyroid-stimulating hormone (TSH) pathway, myo-Ins depletion is also associated with a condition of hypothyroidism and thyroid dysfunctions [177]. Lastly, it is relevant to point out that the altered inositol concentrations may depend on the assumption of pharmacological medicaments, especially antiepileptic drugs (AEDs) like valproate and carbamazepine, or pharmaceuticals for treating bipolar disorder, such as lithium. The peripheral depletion of myo-Ins and consequently also of D-chiro-Ins, due to the chronic use of these substances, may induce symptoms related to PCOS, including an altered endocrine and metabolic unbalance typically correlated with altered inositols’ metabolism in these patients [178]. On these premises, one can figure out how altered inositol levels (or metabolism) impact on several reproductive, hormonal, metabolic, and neurological disorders. Therefore, according to the physiological role of inositols and the pathological implications of altered myo-Ins-to-D-chiro-Ins ratios, inositol therapy should be designed with two different aims: on the one hand, restoring an inositol physiological ratio and, on the other hand, altering this ratio in a controlled manner to achieve specific effects.



A clinical guide to inositols

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A clinical guide to inositols

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Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders Evanthia Diamanti-Kandarakisa, Olga Papaloub, and Christophe O. Soulagec,d a Department of Endocrinology and Diabetes, Professor of Endocrinology, Hygeia Hospital, Athens, Greece Department of Endocrinology and Diabetes, Hygeia Hospital, Athens, Greece c University of Lyon, CarMen Lab, INSERM 1060, INRAE U1397, Lyon, France d Universite Claude Bernard Lyon 1, Lyon, France b

Introduction Industrialization, technological advancements, and their concomitant environmental effects, as well as the rapidly rising prosperity of the “westernized” societies, have given birth to the plague of 21st century, namely, metabolic disease. Through its two main clinical expressions, obesity and type 2 diabetes mellitus (T2DM), metabolic disease is more prevalent than ever, posing one of the greatest threats to health, due to its chronic consequences and its detrimental effects in quality of life and human longevity [1]. In order to be able to limit its burgeoning course, understanding the pathophysiology of metabolic disease has been acknowledged as a global priority. Human energy metabolism is regulated by an incessant hormonal interplay, coordinating food intake and energy expenditure, via metabolically active organs and tissues, such as liver, pancreas, adipose tissue, brain, gut, and thyroid. Genetic predisposition, environmental factors acting as early as during gestation, and lifestyle parameters define the “metabolic identity” of an individual, via their effects in energy homeostasis. As the knowledge base underpinning metabolic disease continues to expand with novel pathophysiological targets being continuously discovered, treatment options should also expand, offering hope for tackling this epidemic in the future. In this context, inositol has been introduced during the past 20 years as a physiological modulator of insulin signaling and glucose metabolism. Moreover, the ratio of its metabolites, myo- and D-chiro-inositol, is crucial for the healthy state of organs and tissues, while an imbalance in their levels or their peripheral tissue depletion may account for pathological conditions, including metabolic disease. Therefore, there was a rising scientific interest toward inositols’ potential implication in the therapeutic management of metabolic disorders. Until now, literature data are rather encouraging, but more consolidated evidence is necessary to establish their therapeutic efficacy in large-scale studies [2]. A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00005-4

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A clinical guide to inositols

The global “metabolic epidemic”: Common soil for a clustering of disorders Prevalence and clinical manifestations Metabolic disease constitute a pathogenetically and clinically heterogeneous entity, which has been highlighted as a major public health emergency during the past 50 years, due to its accompanying multimorbidity. Abdominal obesity, dyslipidemia, hypertension, nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, and T2DM formulate the “constellation” of metabolic disorders (see Fig. 1), all of which have been intimately correlated with an increased risk for a clustering for chronic diseases, such as cardiovascular disease (CVD), hypertension, cancer, neurological diseases, and polycystic ovary syndrome (PCOS) [3]. Obesity and T2DM are considered to be its most prevalent denominators, currently affecting hundreds of millions of individuals of all ages globally and projected to increase during the next decade. Specifically, more than 800 million people worldwide are obese, while simultaneously childhood obesity is expected to skyrocket by 60% in the upcoming decade, reaching almost 250 million by 2030 [4]. Likewise, an exponential increase has been documented in people diagnosed with diabetes mellitus (DM), rising from 108 million in 1980 to 462 million in 2017, corresponding to 6.28% of the world’s total population [5]. The “constellation” of metabolic disorders Obesity

Metabolic syndrome


Risk for: • CVD • Hypertension • Cancer • Neurological diseases • PCOS


NAFLD Genetic predisposition Lifestyle Environmental factors

Fig. 1 The “constellation” of metabolic disorders. Metabolic disease is a pathogenetically and clinically heterogeneous entity, entailing a clustering of clinical conditions, such as abdominal obesity, dyslipidemia, hypertension, nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, and type 2 diabetes mellitus (T2DM), all of which predispose to the development of multiple chronic diseases, including cardiovascular disease (CVD), hypertension, cancer, neurological diseases, and polycystic ovary syndrome (PCOS).

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders

As anticipated, these dramatic epidemiological data are translated into a substantial disease burden, associated with a greater increase to morbidities and mortalities, reduced quality of life, and, unavoidably, escalated healthcare expenses worldwide. First, as body mass index (BMI) increases, the observed risk for cardiometabolic multimorbidity (i.e., T2DM, coronary heart disease, and stroke) is doubled in overweight and more than tenfold in severely obese people compared with individuals with a healthy BMI [6]. Apart from morbidity, there is no doubt that metabolic disease is a major contributor to mortality. In a recently published study from England and Scotland, it was highlighted that excess adiposity now accounts for more deaths than smoking among middle- and oldaged individuals [7]. Furthermore, in 2019, diabetes was the ninth leading cause of death with an estimated 1.5 million deaths directly caused by diabetes [5]. The ongoing COVID-19 pandemic has also unraveled the underlying poor metabolic health in our society and its huge public health burden. There is strong evidence that people with metabolic disorders are not only more susceptible to severe COVID-19 infection and worse outcomes, but also to an increased risk of post-acute sequelae of COVID-19 [8]. For instance, in a recently published study, among 46,441 patients hospitalized with COVID-19, metabolic syndrome was associated with an increased risk of intensive care unit (ICU) admission (adjusted odds ratio [aOR], 1.32), invasive mechanical ventilation (aOR, 1.45), acute respiratory distress syndrome (ARDS) (aOR, 1.36), and mortality (aOR, 1.19), as well as prolonged hospital and ICU length of stay [9]. Concerning hospitalizations, it was estimated that approximately 20.5% of COVID-19 hospitalizations in USA were attributable to diabetes mellitus, 30.2% to obesity (body mass index 30 kg/m2), 26.2% to hypertension, and 11.7% to heart failure, which jointly account for over 500,000 COVID-19 hospitalizations [10]. Not surprisingly, if we try to translate all the above into financial data, numbers are equally disappointing. According to CDC, the estimated annual medical cost of obesity in the United States was $147 billion in 2008 [11] and expected to exceed US$1 trillion by 2025 [4]. Likewise, the economic burden of T2DM is staggering. In a study conducted by the American Diabetes Association (ADA), it was estimated that the total costs of diagnosed T2DM have risen from $245 billion in 2012 to $327 billion in 2017 ($237 billion in direct medical costs and $90 billion in reduced productivity), which represents a 26% increase over a five-year period. Furthermore, it was calculated that individuals with T2DM have approximately 2.3 times higher medical expenditures, compared to the rest of the population [12]. Overall, it becomes clearer than ever that cardiometabolic background is crucial for human well-being and longevity. Combating metabolic disease and its adverse sequelae has arisen in a huge public health challenge, with current efforts and strategies being inadequate to limit its burgeoning course. In the post-pandemic era, metabolic health should be prioritized in healthcare systems globally, in order to effectively take control of the increasing prevalence of metabolic multimorbidity.



A clinical guide to inositols

Pathophysiological components of metabolic disease Metabolic syndrome (MetS) is defined as a cluster of clinical and metabolic abnormalities that directly increases the risk of cardiovascular diseases and T2DM. Insulin resistance, visceral adiposity, atherogenic dyslipidemia, and elevated blood pressure are generally recognized as the main factors that constitute the syndrome. Insulin resistance appears as the cornerstone of MetS formerly often referred to as “insulin resistance syndrome.” It can be defined as an impaired insulin action on its target tissues. Insulin resistance provides a “common soil” connecting apparently unrelated biological/clinical disturbances into a coherent pathophysiological framework. Insulin is a polypeptidic hormone only secreted by the pancreatic β cells whenever glycemia is above its baseline value (>5 mmol/L). The main actions of insulin are to increase glucose uptake in muscle and liver and inhibit hepatic glucose production (through the inhibition of glycogenolysis and gluconeogenesis). Insulin resistance is mainly mediated by post-receptor defects in the intracellular insulin signaling pathway. A major mechanism by which insulin signaling can be negatively regulated is via phosphorylation of certain inhibitory serine residues on insulin receptor substrate 1 and 2 (IRS-1/2). A first unifying hypothesis for the development of insulin resistance is often referred to as “lipotoxicity.” It is now widely admitted that when adipose tissue is absent (lipoatrophy), insulin resistant or overwhelmed by excessive influx of lipids (related to chronic energy imbalance), the triacylglycerol surplus will be deposited at undesirable sites (such as liver, heart, or skeletal muscle), a phenomenon called “ectopic lipid deposition.” The intracellular accumulation of lipids in these organs will promote the production of some toxic lipids species that will in turn trigger the activation of novel protein kinases C (nPKC) with subsequent impairments in insulin signaling via inhibitory serine phosphorylation of IRS proteins [13]. For instance, the accumulation of ectopic lipids yields in the production of diacylglycerols (DAGs) that activate serine kinase such as PKC-θ in muscle and PKC-ε in liver [13]. Ceramides, other lipid species produced in such circumstances, can directly inhibit the phosphorylation of PKB/Akt [14]. Insulin resistance in adipose tissue impairs the anti-lipolytic activity of insulin, leading to an exacerbated lipolysis that increases circulating free fatty acids (FFAs) [15]. A second unifying hypothesis, relating accumulated adipose tissue and the development of insulin resistance, could be inflammation. There is accumulating evidence that inflammatory cytokines (and possibly derived from adipose tissue, such as tumor necrosis factor-α, TNF-α) contribute to the development of insulin resistance [16]. An acute rise of TNF-α in plasma further exacerbates lipolysis, therefore increasing circulatory FFA levels [17]. Inflammatory cytokines such as TNF-α or interleukine-6 (IL-6) induce insulin resistance through the activation of serine/threonine kinases such as Jun N-terminal kinase (JNK), nuclear factor-kappa B (NF-κB), and mammalian target of rapamycin (mTOR) that promote inhibitory serine phosphorylation of IRS-1 [13]. Through the two main mechanisms described above, accumulated fat,

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders

especially visceral adiposity, is thought to be a major contributor to insulin resistance, thus stressing the importance of energy imbalance (i.e., high caloric intake and low energy expenditure) as a pivotal causative factor [18]. For a long time, pancreatic β cells can compensate the poor insulin sensitivity of tissues by oversecreting insulin. The net effect of insulin resistance is therefore the creation of a hyperinsulinemic state that becomes mandatory to maintain euglycemia. In the long run, FFAs and chronic hyperglycemia are also toxic to pancreatic β cells (a phenomenon called “glucolipotoxicity”) and can cause β cell dysfunction, namely, a failure to maintain a high level of insulin secretion. Insulin resistance also contributes to the development of hypertension through the impaired vasodilator effect of insulin [19] and the vasoconstrictive effect of FFAs [20]. Hyperinsulinemia may further increase sympathetic nervous system (SNS) activity and contribute to the development of hypertension [21]. In the context of insulin resistance, the vasodilatory effect of insulin can be lost, while its renal effect on sodium reabsorption remains preserved [21]. Insulin resistance is also known as a major contributor to dyslipidemia. The increased flux of FFAs to the liver stimulates the production of very low-density lipoproteins (VLDL). Under physiological conditions, insulin inhibits the secretion of VLDL into the systemic circulation but this action is impaired when liver becomes insulin resistant. In the setting of insulin resistance, increased flux of free fatty acids to the liver also increases hepatic triglyceride synthesis, leading to hypertriglyceridemia, a hallmark of insulin resistance [22].

Energy homeostasis and glucose metabolism: The emerging role of inositols Inositols are physiologically involved in insulin signaling The main roles of insulin are to promote the cellular uptake of glucose (mainly into adipose and muscle cells) and to inhibit the hepatic production of glucose (issued from glycogen hydrolysis and/or neoglucogenesis) to decrease plasma glucose level. Insulin binds on its target cells onto a tyrosine kinase receptor and triggers its selfphosphorylation onto tyrosine residues located in its cytoplasmic domain [23]. These phospho-tyrosines are recognized by the molecular adaptors IRS-1/2 that also interact with the enzyme phosphatidylinositol 3 kinase (PI3K). IRS-1/2 interacts with the p85 regulatory subunit of PI3K, whose role is to regulate the activity of the p110 catalytic subunit. Activated IRS1/2 promote the phosphorylation of p85, reducing its inhibition of p110 subunit and increasing PI3K enzymatic activity. PI3K promotes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5triphosphate (PIP3) that activates 3-phospho-inositide-dependent kinase 1/2 (PDK1/2) and in turn promotes the serine/threonine phosphorylation of protein kinase B/Akt (PKB/Akt). In the muscle and adipose tissue, activated PKB/Akt will stimulate the translocation of glucose transporters GLUT4 to the plasma membrane to allow glucose to



A clinical guide to inositols

enter into the cells [23]. In the liver, PKB/Akt will trigger the phosphorylation of glycogen synthase kinase 3 (GSK3) to enhance glycogen synthesis, while it will inhibit the transcription factor FOXO1 that promotes gluconeogenesis [23]. Under certain conditions, control of glucose transport by insulin seems to be disconnected from its effect on glycogen synthesis [24,25], suggesting that insulin signaling could proceed along several pathways for glucose transport and glucose metabolism [26]. Larner et al suggested that besides the phosphorylation events initiated by the activation of receptor tyrosine kinase, some intracellular second messengers of insulin could also be generated [27]. These putative second messengers of insulin had been historically extracted from insulin-sensitive tissues (e.g., liver or muscle) stimulated with insulin and exhibited insulin-mimetic activities in vitro as well as in vivo [27]. Myo-inositol (MI) and D-chiro inositol (DCI) are major components of these second messengers released upon the activation of the insulin signaling pathway. These second messengers generated in response to insulin are referred to as “inositol phosphoglycans” (IPGs) [28]. Structural analysis of isolated insulinmimetic IPGs from mammalian insulin-target tissues revealed two different kinds of IPGs [29]. Type-A IPGs are composed of MI and D-glucosamine, while type-P IPGs are composed of D-galactosamine and 3-O-methyl-DCI (i.e., the 3-O-methylated form of DCI). These two classes of IPGs further exhibit different biological activities. While IPG-As mimic the lipogenic activity of insulin in adipose tissue and inhibit cAMP-dependent protein kinase (PKA) [29], IPG-Ps mimic the glycogenic effect of insulin on muscle and liver through the stimulation of pyruvate dehydrogenase phosphatase (PDHP) [29–31]. Type-A IPGs are related to the glycosylphosphatidylinositol (GPI) glycolipids, a major component of GPI anchors found at the surface of every mammalian cells. The origin of type-P IPGs in mammalian cells, however, remains unclear. Insulin is thought to stimulate the cleavage of GPI anchors by some phospholipase C (PLC) and/or phospholipase D (PLD) with the production of free IPGs outside the cells [32]. IPGs are then transported into the cells by an unknown energy-requiring process transporter [33]. The IPGs activate cytosolic phosphoprotein phosphatase 2Cα (PP2Cα) [34]. PP2C in turn dephosphorylates and activates GS as well as phosphoinositide-3 kinase (PI3K) [35]. The activated PP2Cα further stimulates glycogen synthase by both direct (GS) and indirect mechanisms, through the PI3K/Akt pathway. IPGs stimulate pyruvate dehydrogenase (PDH) activity via the activation of pyruvate dehydrogenase phosphatase (PDHP) [36,37] and thus enhance oxidative glucose metabolism through the tricarboxylic acid cycle [27] (Fig. 2). The specific biological activities of several structurally defined insulin-mimetic IPGs were reviewed and compiled in Goel et al. [38]. To get more insight into the structure-activity relationship of IPGs, Hecht et al. [39] chemically synthesized a bunch of IPGs that were extensively tested both in vitro and in vivo (phosphorylation of PKB/Akt, glucose uptake, lipogenesis assay, hypoglycemic activity). In contrast to the previous report, none of the synthetic IPGs tested were insulin-mimetic, questioning their actual pharmacophore and/or biological activity.

Fig. 2 Inositol phosphoglycans (IPGs) as second messengers of insulin. In the canonical insulin signaling pathway, upon binding to its receptor (IR), IR autophosphorylates, recruits, and activates adaptor protein IRS-1. Activated IRS1 activates PI3K to generate PIP3 that in turn activates PDK1 and subsequently PKB/Akt. Activated PKB/Akt triggers the translocation of glucose transporters GLUT4 to plasma membrane to allow extracellular glucose to flow into the cells (in muscle and adipose cells). In muscle and liver cells, PKB/Akt inhibits GSK3 to promote glycogen synthesis. According to the theory of Larner and Brautigan, activation of IR might also be coupled to a heterotrimeric G protein that could activate a phospholipase C or D responsible for hydrolysis of GPI with the release of soluble inositol phosphoglycans (IPGs) outside the cell. Through an unknown transporter, IPGs are imported into the cells where they behave as insulin second messengers. In the cytoplasm, IPGs activate PP2Cα that contributes to the activation of glycogen synthase (GS). In the mitochondria, IPGs activate PDHP to enhance PDH activity and promote glucose oxidative metabolism. Note that all the molecular events presented on this figure may occur in different cell types (e.g., glucose uptake in adipose and muscle cells, glycogen synthesis in liver and muscle cells). GLUT-4, glucose transporter 4; GPI, glycosylphosphatidylinositol; GS, glycogen synthase; GSK3, glycogen synthase kinase 3; IR, insulin receptor; IPGs, inositol phosphoglycans; IRS1, insulin receptor substrate 1; PDH, pyruvate dehydrogenase; PDHP, pyruvate dehydrogenase phosphatase; PDK-1, 3-phosphoinositide-dependent kinase 1; PI3K, phosphoinositide-3-kinase; PIP3, phosphatidylinositol 4,5 bisphosphate; PKB/Akt, protein kinase B/Akt; PP2Cα, phosphoprotein phosphatase 2C alpha. Modified from Croze, M. L., Soulage, C. O. (2013). Potential role and therapeutic interests of myo-inositol in metabolic diseases. Biochimie, 95(10), 1811–1827. https://doi.org/ 10.1016/j.biochi.2013.05.011.


A clinical guide to inositols

Further experimental studies are, however, needed to better understand and document the biological activity of IPGs as intracellular mediators of insulin effects. A latter category of inositol involved in insulin signaling is the family of inositol phosphates. Inositol phosphates are a diverse group of signaling molecules in which hydroxyl groups positioned around an inositol ring are phosphorylated in different combinations by several specific inositol phosphate kinases. They include inositol triphosphate (IP3), inositol pentaphosphate (IP5), inositol hexaphosphate (IP6 also known as phytic acid), and inositol pyrophosphate (IP7). Inositol pyrophosphate (IP7), which is produced in response to insulin, was shown to inhibit PKB/Akt [40], thus contributing to a feedback loop regulating insulin signaling.

Inositols are involved in insulin secretion by pancreatic β cells Besides their effect on insulin signaling, DCI containing IPGs (DCI-IPGs) stimulate insulin secretion from pancreatic β cells [41] through the closure of ATP-sensitive potassium channels. Indeed, the effect of DCI-IPG on insulin secretion is prevented by the pharmacological blockade of ATP-sensitive potassium channels using tolbutamide. The effect of DCI-IPGs on ATP-sensitive potassium channels is also mediated by protein phosphatase 2C (PP2C) since genetic silencing of PP2C gene prevented this effect [41]. Inositol pyrophosphate (IP7) was also shown to promote insulin secretion by pancreatic β cells [42].

Molecular mechanisms and metabolic effects: Experimental and animal data There is accumulating experimental evidence that inositols or inositol derivatives could improve glucose metabolism and glycemic control (reviewed in Ref. [43]). Most experimental studies focused on a handful of compounds, mainly MI, DCI, and D-pinitol (DPI). Myo-inositol (MI) The hypoglycemic action of dietary MI was originally reported by Ortmeyer et al. in Rhesus monkey with type 2 diabetes [44,45]. MI (0.9 and 1.2 g/kg bw) improves glucose tolerance in mice mainly through an insulin-sensitizing effect [46] and increases the translocation of GLUT4 to plasma membrane [47]. MI also seems to be efficient in several animal models of diabetes although some conflicting data exist. MI (1% in diet, in addition to some vitamins) improves insulin sensitivity in pregnant db/+ mice taken as a model of gestational diabetes [48]. MI (1 g/kg bw) decreases intestinal glucose absorption and increases muscle glucose uptake in normal and type 2 diabetic rats [49]. MI (25–50 mg/kg bw) improves glycemic control and dyslipidemia in rats with type 2 diabetes [50]. In human endothelial cells, MI (up to 1 mmol/L) is insulin-mimetic and stimulates the insulin signaling pathway as evidenced by phosphorylation of PKB/Akt [51].

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders

Oral MI supplementation (50 mg/kg bw) alleviates insulin resistance (through the suppression of phospho-STAT3 and IL6 signaling) in a rat model of polycystic ovary syndrome (PCOS) [52]. MI supplementation (0.4 to 10 mg/day) during lactation improves metabolic health and insulin resistance in male rats exposed to inadequate fetal nutrition and diabetogenic diet in adulthood, while surprisingly, no effect was noticed in females [53]. In contrast to these data, MI (1% in diet) failed to improve glycemic control in type 1 diabetic rats [54] or administered at 0.58 g/kg bw it failed to improve insulin resistance in mice fed an high-fat diet [55]. However, this latter dosage could be too low to produce significant effects. D-Chiro-inositol

(DCI) DCI exhibits even more potent insulin-mimetic and insulin-sensitizing activities than MI [27,56–58]. In human endothelial cells, DCI (up to 1 mmol/L) is insulin-mimetic and stimulates the phosphorylation of PKB/Akt [51]. Infusion of chiro-inositol glycan normalizes plasma glucose in type 1 diabetic rat [59], while DCI (15 mg/kg bw) per se decreases plasma glucose in type 2 diabetic rats [60]. DCI (30–60 mg/kg bw) improves insulin-mediated glucose uptake in rats with type 2 diabetes [61]. Dietary administration of DCI to diabetic obese KK-A(y) mice lowers plasma glucose, improves glucose tolerance, and increases insulin pancreas content [62]. In a transcriptomic analysis in primary hepatocytes incubated with palmitate to induce insulin resistance, DCI (10 μmol/L) altered the pattern of expression of genes specifically related to insulin resistance and glucose metabolism [63]. DCI (35 and 70 mg/kg bw, per os) significantly improves glucose metabolism and prevents glucotoxicity (e.g., production of advanced glycation end-products) in db/db diabetic mice [64]. DCI improves insulin sensitivity by the inhibition of hepatic gluconeogenesis and decreased hepatic glucose production (through the stimulation of PKC-ε-IRS/PI3K/AKT signaling pathway) in mice fed a high-fat diet [65]. D-Pinitol

(DPI) DPI (or 3-O-methyl-chiro-inositol) is a methylated derivative of DCI commonly found in plants and vegetables (i.e., a significant dietary source of DCI). Administered to mice, DPI (2 g/kg ip) stimulates the translocation of GLUT4 to plasma membrane in skeletal muscle [47]. DPI (5 mg/kg bw) decreased plasma glucose in type 2 diabetic rats induced by low-dose streptozotocin [60]. DPI activates the insulin signaling pathway (PI3K/Akt pathway) in hypothalamus and could therefore be useful to prevent insulin resistanceassociated brain disorders [66]. In obese mice, DPI enhances glucose-induced insulin secretion but promotes hepatic lipogenesis and triglyceride deposition in liver [67]. In rats, orally administered DPI decreases insulin secretion through upregulation of ghrelin (a known inhibitor of insulin secretion) and inhibits hepatic production of glucose [68].



A clinical guide to inositols

Translating the pathogenetic background into a putative therapeutic role of inositols in metabolic disease Targeting obesity Despite the significant progress made in understanding the multifactorial pathogenesis of obesity, contemporary therapeutic advancements have not been a quantum leap in the clinical management of the disease. Just like its etiology, managing obesity is characterized by a series of therapeutic pillars, including diet, physical exercise, pharmacological treatment, and surgery, targeting different pathophysiological critical points toward generating a negative balance between energy consumption and expenditure. Additionally, the marked variability in the response of individuals to any form of treatment is one of the unique characteristics and unexplained issues of obesity that renders personalized management and combination therapy an imperative need [69]. Due to all its abovementioned physiological roles in energy metabolism and insulin signaling, inositol supplementation has attracted the scientific interest regarding their putative effects in weight management and insulin function [70]. Primary data originated from animal models, in which inositol hexakisphosphate kinase (IP6K)-knockout mice displayed reduced body weight, with decreased percentage of fat mass and concomitant increased percentage of lean body mass. Furthermore, when high-fat diet was administered, IP6K-knockout mice were resistant to obesity and characterized by an improved metabolic profile, including decreased blood glucose, total cholesterol, triglycerides, liver function and leptin, which was attributed to reduced Akt pathway [40,71,72]. Akt is a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways, which were abnormally expressed in skeletal muscle, white adipose tissue, and liver of mice with a targeted deletion of IP6K1, limiting insulin resistance and adipogenesis and serving as a potential therapeutic target for obesity and diabetes [40] (see Fig. 3). White adipose tissue (WAT) remodeling and energy regulation are crucial to obesity pathogenesis. WAT responds to caloric excess through a healthy or unhealthy expansion. Healthy expansion through adipocyte hyperplasia protects against the metabolic complications of obesity. On the other hand, unhealthy expansion through adipocyte hypertrophy is accompanied by detrimental alterations in cell metabolism, including increased lipolysis and leakage of FFAs, impaired insulin signaling partly due to lipotoxicity, increased inflammation, altered adipokine secretion, and ectopic fat deposition [73]. Thus, understanding adipose tissue physiology and its contribution to systemic energy homeostasis, as well as therapeutical targeting it, is essential in tackling obesity and its metabolic comorbidities. Toward this approach, the role of inositol in adipose tissue metabolism was recently evaluated in an experimental study using a human adipocyte cell line. In that study, the authors found that DCI was actively involved in the differentiation and normal function of human adipocytes where it synergizes with insulin and estrogens

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders


Akt/ GSK3β/ mToR pathway



↓Weight gain ↓Insulin resistance ↓Adipogenesis ↓TCHOL, ↓TGL, ↑HDL Hepatoprotecon in NAFLD ↓Energy storage ↓Lipid accumulation ↑Adipocyte browning gene expression (PGC1, PRDM16, PPARα)


Ins/Est signal transducon pathways

Mainly in depleted individuals (prediabetes, T2DM, PCOS) MI:

↑Expression acvaon IRS1/GLUT4 ↑Differentiation of adipocytes ↓Fasting Ins/Glu/HOMA-IR/HbA1c Improvement of lipid profile/BP/BMI ↓GDM rates/ Ins Tx in GDM/ ↑fetal benefits Improvement of gut microbiota ?Diabec nephropathy

Fig. 3 Summary of inositols’ actions in metabolic disorders. Inositols seem to be promising in targeting most aspects of metabolic disorders. Possible mechanisms are also shown. Hexakisphosphate kinase (IP6K1), AMP-activated protein kinase (AMPK), total cholesterol (TCHOL), triglycerides (TGL), highdensity lipoprotein (HDL), nonalcoholic fatty liver disease (NAFLD), uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1), PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor alpha (PPARα), insulin (Ins), estrogen (Est), insulin receptor substrate-1 (IRS1), glucose transporter type 4 (GLUT4), type 2 diabetes mellitus (T2DM), polycystic ovarian syndrome (PCOS), glucose (Glu), Homeostasis model Assessment of Insulin Resistance (HOMA-IR), glycosylated hemoglobin (HbA1c), body mass index (BMI), gestational diabetes mellitus (GDM), treatment (Tx).

through the recruitment of signal transduction pathways involved in lipid and glucose storage, increasing the expression and activation of IRS1 and GLUT4 [74]. Likewise, IP6K1 deletion in mice fat cells increases energy expenditure and lipid oxidation via the AMP-activated protein kinase (AMPK) and decreases fatty acid synthesis in 3T3L1 adipocytes [75]. Finally, inositol seems to be also involved in WAT browning, an innate physiological process that results in increased cellular thermogenesis and energy expenditure. Specifically, IP6K1 knockout in adipocytes led to increased oxygen consumption rate and adipocyte browning genes expression, such as uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1), PR domain containing 16 (PRDM16), and peroxisome proliferator-activated receptor alpha (PPARα), indicating that IP6K1 deletion might enhance the browning of adipocytes [76].



A clinical guide to inositols

Another recently discovered, novel player in obesity pathogenesis is gut microbiota, the collective community of microorganisms in the gastrointestinal tract. Apart from their pure gastrointestinal role, gut microbiome composition is crucial in altering metabolic processes via various mechanisms. For instance, a high abundance of bacteria that ferment carbohydrates leads to increased rates of short-chain fatty acid (SCFA) biosynthesis, providing an extra source of energy for the host, which is eventually stored as lipids or glucose. Furthermore, microbiota can aggravate low-grade inflammation, and consequently, insulin resistance, via increasing intestinal permeability to bacterial lipopolysaccharides (LPS) [77,78]. Although data are still extremely limited, it seems that MI intake may have a metabolic role in concert with gut microbiota. In a study with rats fed a highsucrose diet, MI administration normalized hepatic triglycerides concentration, suppressed the hepatic expression of lipogenic genes, through a concomitant improvement in microbiota composition [79,80]. Despite experimental data being promising so far, current literature lacks robust human data to support the role of inositol in obesity. As far as we know, the only human studies with inositol’s effects on weight mainly come from women with polycystic ovary syndrome (PCOS) [81]. Simultaneously, there are some preliminary clinical reports providing compelling evidence supporting the beneficial effects obtained with inositol in insulin-resistant patients that may be associated with beneficial clinical outcomes. However, it is rather imperative to proceed to well-organized, randomized clinical trials that will let us clearly establish inositol’s challenging role in obesity management.

Targeting insulin resistance and type 2 diabetes mellitus As clearly mentioned above, inositol, through its isomers (MI and DCI), and probably some of its phosphate intermediate metabolites and correlated enzymes (like IP6K1) are actively implicated in insulin signaling and glucose metabolism, by influencing distinct pathways [82]. Experimental and clinical data highlight that impairment of physiological processes relating to MI uptake, de novo biosynthesis, phosphoinositide-cycle regeneration, efflux, and degradation, which can result in MI depletion in the insulinsensitive tissues, can predispose individuals to insulin resistance and T2DM. Simultaneously, decreased epimerase activity and a reduction of MI re-absorption at the kidney level are also observed in diabetic individuals or in siblings from families with predisposition to T2DM, further implicating inositol in the multifactorial etiology of metabolic disease [79,82]. Currently, the most robust data regarding the insulin-mimetic properties of inositol originate from women with PCOS [83]. Unlike other tissues in T2DM and PCOS that show an increased insulin resistance, such as the liver and muscle, the ovary remains sensitive to insulin. As a result, the subsequent hyperinsulinemia possibly leads to an overepimerization of MI to DCI, via the insulin-dependent NAD/NADPH epimerase

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders

enzyme in the ovarian tissue, supporting the “ovarian paradox” hypothesis [83]. The increased availability of DCI as opposed to the depletion of MI and the worsening features of PCOS when DCI only was administered, led to the suggestion that both isoforms should be administered aiming to restore the deranged imbalance of MI/DCI. After trials with various combinations of MI/DCI administered to an androgenic-like experimental model of female mice, the daily treatment with 420 mg/kg MI/DCI in a 40:1 ratio seemed to be the most efficient in reversing the PCOS phenotype [84]. Apart from their reproductive effects, inositol isoforms were efficacious in improving insulin resistance, with a significant reduction in glucose, C-peptide levels, and HOMAIR index; decreasing blood pressure; and improving their lipid profile. By reducing insulin resistance, inositol can ultimately ameliorate hyperandrogenemia, and improve menstrual regularity and fertility rates [85]. In this context, a series of experimental and interventional studies conducted in both humans and animals tried to elucidate if this insulin-sensitizing role of inositol can be observed in metabolic disease and T2DM. More specifically, MI supplementation in rats inhibited intestinal glucose absorption and increased muscle glucose uptake [49], while a DCI-derivative glycan originally isolated in beef liver functioned as an insulin-mimetic and sensitizer, decreasing body weight and food intake when administered intracerebroventricularly in mice [86]. Likewise, human studies support that MI supplementation, with or without D-chiro-inositol supplementation, at doses ranging between 2 and 10 g/day can improve insulin sensitivity, lipid profile, and arterial blood pressure in people with prediabetes or metabolic syndrome. In a recent study of patients with T2DM with suboptimal glycemic control (HbA1c 7.0%–10.0%) already in therapy with glucose-lowering agents, the supplementation of MI and DCI in their regular therapeutic regimen for 3 months resulted in a significant reduction both in fasting glucose (192.660.2 versus 160.936.4; P ¼0.02) and in HbA1c (8.6% 0.9% versus 7.7%0.9%; P ¼0.02) compared to baseline, devoid of changes in arterial blood pressure, lipid profile, and BMI [87]. Analogous conclusions were reached by the authors of a recent meta-analysis and systematic review, which evaluated a total of 20 RCTs with 1239 subjects. MI given at doses of 600–4000 mg was accompanied by a reduction in fasting plasma glucose, post-OGTT plasma glucose, fasting insulin, and HOMA-IR, independently from changes in body weight [88]. Finally, due to the observation that there is an increased loss of MI in urine in patients with insulin resistance and T2DM, a potential further application of MI in diabetic nephropathy is being evaluated, but adequate human trials are still lacking [82,89]. Unsurprisingly, the effectiveness of MI has been expanded also in gestational diabetes mellitus (GDM) prevention and treatment. Several clinical studies have demonstrated its effectiveness in lowering GDM rates, improving gestational glycemia, lipid and insulin resistance parameters, as well as in reducing the need for insulin therapy should GDM develop later [2]. Furthermore, a recent study by D’Anna et al. emerged that MI plus



A clinical guide to inositols

α-lactalbumin supplementation had also beneficial effects for the fetus, namely, reduced fetal abdominal circumference and neonatal subcutaneous adipose tissue thickness, while no cases of preterm birth occurred in the treated group, compared with 15.2% observed in the controls [90]. Overall, there is no doubt that inositol does have a physiological and pathophysiological role in insulin signaling and glucose metabolism. What is left to be done now is to translate these observations into substantial clinical benefits. Although there are some preliminary clinical reports, there is need for more compelling evidence in order to introduce inositol in our “therapeutic quiver” against metabolic disease.

Targeting other aspects of metabolic syndrome Dyslipidemia constitutes an integral part of MetS, contributing equally to the development of CVD. With insulin resistance being the orchestrator, dyslipidemia of MetS is characterized by the so-called lipid triad—high levels of plasma triglycerides, low levels of high-density lipoprotein (HDL) cholesterol, and the appearance of small, dense, lowdensity lipoproteins (sdLDL)—and excessive postprandial lipemia, all of which originate from hepatic overproduction of large triglyceride-rich very low-density lipoproteins (VLDL) [91]. In a recent study with a large sample of overweight/obese adults with T2DM, it was observed that the presence of the abovementioned lipid abnormalities was associated with higher risks of atherosclerotic CVD events, highlighting that tackling dyslipidemia is crucial in limiting the adverse effects of atherosclerosis [92]. In this context, a plethora of studies have been conducted to determine whether inositol supplementation had any effect on lipid profile, mainly in individuals with MetS or PCOS [79]. In a study of 80 postmenopausal women affected by metabolic syndrome, MI supplementation resulted in a decrease in triglycerides and an increase in HDL levels, along with a decrease in diastolic blood pressure and HOMA index [93]. Similarly, in a study from Korea with 30 patients with T2DM soybean-derived pinitol (D-3-O-methyl-chiro-inositol) administration led to decreased total cholesterol, LDL, and LDL/HDL ratio and increased HDL, but did not affect triglyceride levels [94]. Finally, in a meta-analysis of a total of 14 randomized controlled trials (RCTs) conducted in populations with metabolic diseases, it was highlighted that inositol supplementation is accompanied by an improvement in triglycerides, total and LDL cholesterol levels, but has no apparent effects on HDL-cholesterol levels [95]. Unsurprisingly, beneficial effects have also been observed in the PCOS population [96]. Constantino et al. studied the effects of MI plus folic acid in 42 women with PCOS and showed that plasma triglycerides significantly decreased, compared to placebo, in parallel with an improvement of insulin sensitivity [97]. More recently, Shokrpour et al. compared the effects of 12-week MI and metformin administration on glycemic control, lipid profile, as well as gene expression related to insulin and lipid metabolism

Effectiveness of Myo- and D-chiro-inositol in the treatment of metabolic disorders

in women with PCOS, and displayed beneficial effects on triglycerides and VLDLcholesterol levels, without, however, modifications of LDL, HDL, or LDL receptor expression [98]. Overall, it is clear that inositols can beneficially affect lipid abnormalities, as a result of various mechanisms, including insulin resistance amelioration or visceral fat and hepatic lipid accumulation decrease. Large-scale RCTs are needed to validate all the above data and establish inositols as a mainstay in the therapeutic management of dyslipidemia. In concert with the increase in prevalence rates of obesity and MetS, the prevalence of NAFLD has increased dramatically to over 25% of the population worldwide, which rises over 60% in high-risk populations, like patients with T2DM. NAFLD is defined by the accumulation of intracellular fat in >5% of hepatocytes on imaging or histology, in the absence of other causes of hepatic steatosis such as excessive alcohol intake, certain metabolic conditions, or drug use, and is considered to be the hepatic component of MetS, also contributing to comorbidities, including T2DM and CVD and resulting in a large healthcare burden. The pathogenesis of NAFLD is complex. However, insulin resistance and adipose tissue dysfunction stand centrally, causing an increased flux of circulating FFAs to the liver and altering hepatic metabolism, toward increased hepatic gluconeogenesis, hepatic insulin resistance, and hyperglycemia [99]. Given its role in other metabolic disorders, it is rather challenging to unravel whether inositol has also a beneficial effect in NAFLD. Overall, current literature data are limited, with the majority of them originating from animal studies. However, it was observed that inositol deficiency is associated with increased fatty liver, while simultaneously inositol supplementation in animal models of fatty liver resulted in the reduction of hepatic triglycerides and cholesterol accumulation and maintenance of a normal ultrastructural liver histopathology [100]. Likewise, in the only human study of existing literature, in which 90 subjects with ultrasonography-proven NAFLD were randomly assigned to the placebo, low-dose (300 mg/d), or high-dose (500 mg/d) of pinitol for 12 weeks, it was found that pinitol significantly reduced liver fat, postprandial triglycerides, AST levels, and lipid peroxidation by increasing glutathione peroxidase activity. All these modulatory effects upon energy and metabolic pathways, along with the amelioration of oxidative stress and fatty acid accumulation, can exert hepatoprotective benefits in NAFLD subjects [101].

Conclusions Inositol and its two major isoforms are considered physiological components and second messengers of various biological processes, including insulin signaling and glucose homeostasis. Any tissue deficit or potential perturbation of inositol physiology can be translated in a disruption of insulin signaling in metabolically active organs, development of insulin resistance, and ultimately manifestation of metabolic disease. Thus, inositol is



A clinical guide to inositols

reasonably acknowledged as potent target in tackling the contemporary epidemic of obesity and T2DM. Undoubtedly, there is a need for more well-organized and large-scale studies to validate our experimental and clinical observations and further elucidate the role of inositol in human metabolism. However, inositol has been triumphantly introduced in the field of obesity and metabolic disease and continues to gain reputation, due to its efficacy, simple administration, safety, and compliance.

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[86] Jeon Y, Aja S, Ronnett GV, Kim E-K. D-chiro-inositol glycan reduces food intake by regulating hypothalamic neuropeptide expression via AKT-FoxO1 pathway. Biochem Biophys Res Commun 2016;470(4):818–23. https://doi.org/10.1016/j.bbrc.2016.01.115. [87] Pintaudi B, Di Vieste G, Bonomo M. The effectiveness of myo-inositol and D-chiro inositol treatment in type 2 diabetes. Int J Endocrinol 2016;2016:9132052. [88] Min˜ambres I, Cuixart G, Gonc¸alves A, Corcoy R. Effects of inositol on glucose homeostasis: systematic review and meta-analysis of randomized controlled trials. Clin Nutr (Edinburgh, Scotland) 2019;38(3):1146–52. https://doi.org/10.1016/j.clnu.2018.06.957. [89] Sharma I, Tupe RS, Wallner AK, Kanwar YS. Contribution of myo-inositol oxygenase in AGE: RAGE-mediated renal tubulointerstitial injury in the context of diabetic nephropathy. Am J Physiol Ren Physiol 2018;314(1):F107–21. https://doi.org/10.1152/ajprenal.00434.2017. [90] D’Anna R, Corrado F, Loddo S, Gullo G, Giunta L, Di Benedetto A. Myoinositol plus α-lactalbumin supplementation, insulin resistance and birth outcomes in women with gestational diabetes mellitus: a randomized, controlled study. Sci Rep 2021;11(1):8866. https://doi.org/10.1038/s41598-02188329-x. [91] Adiels M, Olofsson S-O, Taskinen M-R, Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipidemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2008;28 (7):1225–36. https://doi.org/10.1161/ATVBAHA.107.160192. [92] Kaze AD, Santhanam P, Musani SK, Ahima R, Echouffo-Tcheugui JB. Metabolic dyslipidemia and cardiovascular outcomes in type 2 diabetes mellitus: findings from the look AHEAD study. J Am Heart Assoc 2021;10(7), e016947. https://doi.org/10.1161/JAHA.120.016947. [93] Giordano D, Corrado F, Santamaria A, Quattrone S, Pintaudi B, Di Benedetto A, D’Anna R. Effects of myo-inositol supplementation in postmenopausal women with metabolic syndrome: a perspective, randomized, placebo-controlled study. Menopause (New York, NY) 2011;18(1):102–4. https://doi. org/10.1097/gme.0b013e3181e8e1b1. [94] Kim J-I, Kim JC, Kang M-J, Lee M-S, Kim J-J, Cha I-J. Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: a randomized controlled study. Eur J Clin Nutr 2005;59(3):456–8. [95] Tabrizi R, Ostadmohammadi V, Lankarani KB, Peymani P, Akbari M, Kolahdooz F, Asemi Z. The effects of inositol supplementation on lipid profiles among patients with metabolic diseases: a systematic review and meta-analysis of randomized controlled trials. Lipids Health Dis 2018;17(1):123. https://doi.org/10.1186/s12944-018-0779-4. [96] Unfer V, Facchinetti F, Orru` B, Giordani B, Nestler J. Myo-inositol effects in women with PCOS: a meta-analysis of randomized controlled trials. Endocr Connect 2017;6(8):647–58. https://doi.org/ 10.1530/EC-17-0243. [97] Costantino D, Minozzi G, Minozzi E, Guaraldi C. Metabolic and hormonal effects of myo-inositol in women with polycystic ovary syndrome: a double-blind trial. Eur Rev Med Pharmacol Sci 2009;13 (2):105–10. [98] Shokrpour M, Foroozanfard F, Afshar Ebrahimi F, Vahedpoor Z, Aghadavod E, Ghaderi A, Asemi Z. Comparison of myo-inositol and metformin on glycemic control, lipid profiles, and gene expression related to insulin and lipid metabolism in women with polycystic ovary syndrome: a randomized controlled clinical trial. Gynecol Endocrinol 2019;35(5):406–11. https://doi.org/ 10.1080/09513590.2018.1540570. [99] Ruissen MM, Mak AL, Beuers U, Tushuizen ME, Holleboom AG. Nonalcoholic fatty liver disease: a multidisciplinary approach towards a cardiometabolic liver disease. Eur J Endocrinol 2020;183(3): R57–73. https://doi.org/10.1530/EJE-20-0065. [100] Pani A, Giossi R, Menichelli D, Fittipaldo VA, Agnelli F, Inglese E, Romandini A, Roncato R, Pintaudi B, Del Sole F, Scaglione F. Inositol and nonalcoholic fatty liver disease: a systematic review on deficiencies and supplementation. Nutrients 2020;12(11). https://doi.org/10.3390/nu12113379. [101] Lee E, Lim Y, Kwon SW, Kwon O. Pinitol consumption improves liver health status by reducing oxidative stress and fatty acid accumulation in subjects with nonalcoholic fatty liver disease: a randomized, double-blind, placebo-controlled trial. J Nutr Biochem 2019;68:33–41. https://doi.org/ 10.1016/j.jnutbio.2019.03.006.



Treating PCOS with inositols: Choosing the most appropriate myo- to D-chiro-inositol ratio Arturo Bevilacquaa,b, Simona Dinicolab,c, and Mariano Bizzarrib,c,d a Department of Dynamic, Clinical Psychology and Health Studies, Sapienza University, Rome, Italy The Experts Group on Inositol in Basic and Clinical Research (EGOI), Rome, Italy c Systems Biology Group Lab, Rome, Italy d Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy b

Introduction: PCOS and polycystic ovaries Included among, but not exclusive of, PCOS signs are polycystic ovaries, observed in approximately 20% of women of reproductive age [1]. The diagnosis of polycystic ovaries is made by transvaginal ultrasound, which ascertains the presence of the pathology in either one or both ovaries. Polycystic ovaries display an overall enlargement and presence of several follicles with cortical distribution and similar size (2–9 mm in diameter) and central stromal brightness [2,3]. Although information on human ovarian histology is scarce due to obvious limits in experimental approaches in our species, some features have been observed over the years. These include large follicle size, presence of immature follicles, multiple cystic follicles often covered by a fibrous capsule, absence of corpus luteum due to anovulation, and, at a cellular level, abnormal extension of the luteal component in the follicular compartment or hyperthecosis [4]. To investigate different features and pathophysiology of PCOS, researchers have focused on animal models for over three decades. In particular, rat and mouse models have brought about a considerable body of information on histological features of polycystic ovaries, including cystic follicles with the absence of the oocyte; thin layers of granulosa cells; absence of normal antral follicles; limited numbers of corpus luteum indicating decreased ovulation [5]. As reported [6], early tertiary follicles containing a living oocyte from polycystic mouse ovaries have a hyperplastic theca cell layer. Bevilacqua and colleagues [7] quantified this histological feature by measuring the thickness of the theca cell and granulosa cell layers and calculating their ratios (theca cell/granulosa cell ratio, TGR) in mice under various experimental conditions. TGR ranged from approximately 0.6 in follicles from control ovaries to 1.2 in follicles from polycystic ovaries. Therefore, while the granulosa cell compartment normally expands in control follicles supporting estrogen synthesis, it is substantially

A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00006-6

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A clinical guide to inositols

reduced in favor of the theca cell compartment in polycystic follicles. This is strictly associated with a lack of conversion of androgens into estrogens [8], with a consequent shift toward an androgenic phenotype [9]. Interestingly, the TGR parameter, which unfortunately has been rarely quantified in histological studies, is a reliable predictor of mouse fertility by regression analysis [7].

Mechanisms of action of inositol and its role in PCOS Inositols and glucose metabolism Inositol and inositol-related metabolites have a reknown role in insulin signaling, first demonstrated when it was shown that under insulin stimulation, liver plasma membrane releases inositol phosphoglycans (IPG), containing either myo-inositol (IPG-A) or D-chiro-inositol (IPG-P), through the activation of a phosphatidylinositol-specific phospholipase C [10]. Hence, both myo-inositol (MI) and D-chiro-inositol (DCI) exert an insulin-mimetic activity when incorporated into IPGs, acting as second messengers downstream of insulin receptors [11]. IPG-P, or inositol phosphoglycan-phosphatase stimulator, promotes the activation of pyruvate dehydrogenase phosphatases (PDHP), leading to pyruvate dehydrogenase (PDH) activation [12]. IPG-A, or inositol phosphoglycan-AMP kinase inhibitor, inhibits both protein kinase A and adenylyl cyclase (AC) [13]. Inositol phosphoglycans act as insulin-mimetic factors when administered to normal or diabetic rats, reduce hyperglycemia, and promote glycogenesis [14]. Insulin stimulates the hydrolysis of glycosylphosphatidylinositol (GPI) through direct activation of phosphatidylinositol phospholipase C (PLC) and D (PLD), thus generating water-soluble inositol phosphoglycans [15]. Once insulin activates its receptors, IPGs are released outside the cell membrane and then uptaken through an ATP-dependent inositol-glycan transporter [16]. Inside the cells, IPGs activate cytosolic phosphoprotein phosphatase 2C-α (PP2Cα) and the mitochondrial PDH, thereby enhancing pyruvate dehydrogenase activity and oxidative glucose metabolism along the tricarboxylic acid cycle. In turn, activated PP2Cα stimulates glycogen synthase (GS) either directly or indirectly, through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [17]. Activation of Akt inactivates glycogen synthase kinase-3 (GSK-3)—thus fostering GS activity—and enhances GLUT-4 translocation, promoting glucose uptake. Furthermore, in rats, IPGP inhibits the glucose-stimulated insulin release from pancreatic β-cells, thus suggesting a putative feedback mechanism between insulin and released IPG-P [18]. Overall, these effects point to a general antidiabetic activity triggered by IPGs and, considering the metabolic nature of PCOS, suggest that inositols may significantly attenuate insulin resistance and thus play a relevant role in the pathogenesis of this disease. Insulin also stimulates MI epimerization to DCI, a mechanism that is severely impaired when insulin-sensitive tissues (muscle, fat, and liver) become insulin resistant. Thus, an increased MI-to-DCI ratio is a reliable measure of insulin resistance [19]. Conversely, reduced DCI levels are usually seen in urine and muscle tissue of type 2 diabetic

Treating PCOS with inositols

patients [20]. Additionally, insulin resistance in both type 2 diabetic subjects and healthy controls appears to be linearly correlated with lower DCI levels in urine [21]. Notably, insulin stimulates IPG-P release in normal subjects but not in insulin-resistant PCOS patients [22]. This is a very intriguing result as it suggests that insulin resistance in PCOS may be linked to either a deficiency in membrane-bound IPG or an impaired epimerization of MI to DCI. Moreover, inadequate MI supply negatively affects DCI levels and worsens insulin resistance in PCOS patients [23]. We suppose that reduced MI availability may in turn lead to decreased conversion into DCI, especially in patients in whom high levels of glucose antagonize MI uptake and promote its kidney reabsorption [24]. However, recent findings indicate that IPGs lack insulin-mimetic effects, challenging researchers to reconsider the role of these molecules in insulin signaling [25]. No convincing explanation of such enigma has been proposed, and it can be hypothesized that synthetic IPGs lack some critical cofactor(s) required for the insulin-mimetic activity. Additionally, MI may directly promote the activation of insulin receptor substrate (IRS) and Akt [26], while enhancing GLUT-4 translocation through the cell membrane independently of insulin stimulation [27] or glucose uptake [28]. MI may likely counteract insulin resistance by impairing IP6K1-driven synthesis of IP7. Indeed, overactivation of IP6K1, usually triggered by sustained insulin stimulation, promotes IP7 synthesis and subsequent inhibition of Akt activity by preventing its interaction with PI3K, ultimately reducing insulin sensitivity and protein synthesis via the GSK3β and mTOR signaling pathways [29]. On the contrary, IP6K1-knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Noteworthy, MI specifically downregulates IP6K1 expression, normalizing the PI3K/Akt axis [30].

Inositols and steroidogenesis Increasing evidence suggests that MI, DCI, and inositol phosphates participate in several pathways of gonadal steroidogenesis, not limited to a modulation of insulin resistance [31]. In women, MI is involved in FSH-mediated pathways that regulate proliferation and maturation of granulosa cells. Indeed, MI modulates the FSH-mediated anti-Mullerian hormone (AMH) production, therefore playing a pivotal role in determining oocyte maturation and transport in the oviduct as well as ensuring the good quality of embryos [32]. Ovaries, as well as other tissues, show a specific MI:DCI ratio. DCI levels are higher in tissues that metabolize glucose actively, as expected by the insulin control of the conversion of MI to DCI [33]. Since insulin resistance does not affect ovaries, in the presence of high insulin levels in insulin-resistant patients, an increased conversion of MI into DCI occurs [34]. In turn, high DCI levels may be detrimental for the ovarian function, negatively affecting the quality of oocytes and developing embryos [35]. Moreover, DCI stimulates the ovarian production of androgens by theca cells [36] and decreases the expression of the enzyme aromatase with a reduced conversion of testosterone into estrogens [37]. Thus, an alteration of the MI:DCI ratio may likely explain the imbalance in sex hormones observed in PCOS patients.



A clinical guide to inositols

On the other hand, upon MI treatment, the LH/FSH ratio in the plasma of PCOS patients is significantly decreased [38,39], counteracting the downregulation of FSH and subsequent reduction in granulosa cell aromatase, which represents a hallmark of PCOS [40]. Moreover, within in vitro fertilization protocols, MI supplementation allows to significantly reduce the amount of FSH administered [41].

Clinical and preclinical data Inositols in PCOS treatment Insulin resistance and/or hyperinsulinemia have a recognized role in the pathophysiology of PCOS [42], affirmed over the years, through several in vitro and in vivo studies. Approximately 35% of lean women and 80% of obese women with PCOS also suffer from an insulin signal impairment [43]. Since IPG-P and IPG-A act as second messengers of insulin [10,44,45] and inositol metabolism is severely deregulated in follicle cells of PCOS patients [34], a growing body of research has investigated the therapeutic efficacy of inositols for this pathology. Cheang and colleagues confirmed that an impairment in the inositol phosphoglycan pathway might cause a derangement in the insulin signaling in PCOS [46]. Therefore, the rationale for the use of MI and DCI as a valuable therapeutic approach to PCOS consists primarily in their “insulin-mimetic” action. Treatment with inositols proved to be free from side effects 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) [47]. A 6-month treatment with 2 g MI twice a day was found to effectively restore ovarian activity and fertility in patients with PCOS [48]. In this study, MI reactivated the normal ovulatory activity in 72% of patients with a pregnancy rate of 40% during the 6-month observation period. These findings have been replicated in several studies [39,49]. MI treatment significantly reduced patients’ plasma LH, prolactin, testosterone, insulin levels, and LH/FSH ratio, while HOMA index and insulin sensitivity, expressed as glucose-to-insulin ratio, significantly improved. Moreover, menstrual cycle was restored in all subjects suffering from amenorrhea/oligomenorrhea. Namely, the hormonal profile was significantly improved, while ovulation and regular menses—in both obese and lean women—were restored [50]. Other studies have demonstrated that MI significantly improves many biochemical and clinical parameters related to hyperandrogenism and dysmetabolism of PCOS [38,51–53]. However, the association of MI with DCI is still a matter of debate. Specifically, a consensus still lacks regarding the respective percentage of MI and DCI in treatment protocols, as currently available clinical trials cannot provide definite answers due to their high heterogeneity. Clinical results are difficult to compare, since they derive from studies involving nutraceutical formulas in a wide range of MI:DCI ratios that vary from 0.4:1 to around 100:1. According to current commercial preparations, the daily

Treating PCOS with inositols

dose of DCI, alone or with MI, have low (less than 300 mg/die), medium (300600 mg/die), or high contents (600-1200 mg/die). Some recent reports [54] only consider the improvement in insulin transduction and glucose utilization that inositols exert through their IPGs derivatives [55]. However, these papers do not consider that MI and DCI display opposite effects upon ovary and steroidogenesis. Moreover, as mentioned above, recent findings have shown that DCI can modulate the expression of genes encoding for steroidogenic enzymes in human granulosa cells, reducing the transcription of both aromatase and cytochrome P450 side-chain cleavage mRNAs in a dose-dependent fashion [37]. Furthermore, DCI increases testosterone levels in theca cells from women with PCOS [36]. These data warrant caution in treating PCOS patients with high doses of DCI, as also suggested by a Cheang et al. [46]. On the other hand, while DCI inhibits aromatase, MI can modulate steroidogenesis in the ovary by exerting complex effects upon cytoskeleton architecture [56]. Therefore, it is not surprising that in physiological homeostatic conditions, the MI:DCI ratio in ovarian tissues is kept within the range of 70-100:1, while in ovaries from PCOS patients, this ratio is pathologically decreased [35]. Furthermore, high levels of DCI show detrimental effects upon blastocyst quality [57]. Some clinical studies have recently confirmed these results [58], thus providing a preliminary confirmation of the hypothesis first suggested by Unfer.

The 40:1 approach Several studies showed that MI and DCI can be usefully integrated in the clinical management of PCOS and represent a reliable alternative to conventional treatments for insulin resistance [59–61]. Specifically, a formula comprising 2 g MI and DCI in amounts corresponding to their physiological plasma molar ratio of 40:1 has demonstrated a significant clinical efficacy [58,62]. An appreciable step forward in understanding the tricky interplay between MI and DCI in the ovarian physiology is derived from a recent work in which Bevilacqua and coworkers tested the efficacy of different MI +DCI formulas in PCOS-modeled mice [7]. In this study, the therapeutic effects of various MI and DCI ratios were tested in an experimental mouse model of PCOS. Mice were preliminarily exposed to a continuous light regimen for 10 weeks to induce a PCOS-like phenotype; they were then returned to a normal light/dark cycle and divided in groups, receiving 10-day-long (corresponding to 2.5 ovulatory cycles) treatments with various MI and DCI ratios or water. Formulas tested provided a total amount of 420 mg/kg/day of MI and DCI, corresponding to a human dose of 2 g/day in the respective ratios of 5:1, 20:1, 40:1, and 80:1. While at the end of the inducing period, both uteri and ovaries had clear signs of abnormality, with histological features of PCOS, after 10 days of treatment with the MI and DCI ratio of 40:1, mice displayed normal uteri and ovaries. Moreover, physiological thickness of



A clinical guide to inositols

theca and granulosa cells with a normal TGR, and complete folliculogenesis, including growing and preovulatory stages, were restored. Among the other formulas, MI and DCI 80:1 seemed to be also effective although providing only a partial normalization of uterus and ovaries. Plain water-treated, control mice also showed some but more limited signs of recovery. On the contrary, doses containing higher amounts of DCI were not only ineffective but had toxic effects. Histological analysis relative to MI and DCI 20:1 and 5:1 ratios revealed the presence of scattered primary and secondary follicles, atypical and disorganized ovarian tissues. When the fertility of mice subjected to various recovery treatments was tested, MI and DCI at the 40:1 ratio also displayed the highest efficacy, with pregnancy and delivery times similar to those of healthy control mice. Water- or MI and DCI 80:1-treated PCOS mice had a slower but visible recovery, while mice treated with MI and DCI ratios below the threshold of 40:1, thus containing higher amounts of DCI, displayed prolonged infertility. In conclusion, while confirming the efficacy of the MI and DCI 40:1 ratio at an experimental level, these results further showed that the mammalian ovary well tolerates high doses of MI but is negatively affected by high doses of DCI. To help understand these findings, we should recall that the ovarian MI:DCI molar ratio is maintained around the value of 100:1 in healthy women but drops to 0.2:1 in PCOS patients. Conversely, high levels of DCI, usually occurring in the follicular fluid of patients enrolled in IVF programs, are harmful for oocyte and blastocyst quality [35].

High D-chiro-inositol dosage The above-mentioned results and the observation that DCI reduces aromatase expression in human granulosa cells [37], allowed Bevilacqua and coworkers to hypothesize that, similar to the effects of the aromatase inhibitor letrozole, administration of high doses of DCI to normal female mice would produce either an androgenic PCOS-like condition or other ovarian lesions. In a recent work, this hypothesis was tested by treating juvenile mice with 250, 500, and 1000 mg/kg/day of DCI, corresponding to human doses of 1200, 2400, and 4800 mg/day, respectively [63], for 3 weeks, spanning 5 ovulatory cycles [64]. The lowest 250 mg/kg/day dose of DCI produced morphological signs typical of PCOS, similar to those displayed by mice treated with letrozole, used as a positive control. In addition, in these mice, (i) uteri had the macroscopic aspect typical of non-cycling animals; (ii) the ovulatory cycle was blocked in the majority of them; (iii) serum testosterone levels were significantly increased with respect to untreated mice; (iv) ovarian aromatase expression was decreased. These observations provide the first evidence of a specific downregulation of aromatase mediated by DCI in an in vivo system and confirm previous in vitro observations [37]. Higher doses of DCI were harmful for ovarian tissue organization producing paucity/absence of growing follicles, presence of enlarged follicles with general hyperplasia of follicular and/or stromal cells. This was associated with

Treating PCOS with inositols

minimal serum levels of testosterone and ovarian aromatase, suggesting a blockade in gonadal steroidogenesis. While providing evidence that in the mouse, a daily dosage of 250 mg/kg of DCI for ten days induces an androgenic PCO-like syndrome, these findings deserve full attention for clinical practice. In fact, the PCOS-inducing dose corresponds to a daily dose in the range of 1.0–1.5 g often suggested in therapeutic regimens of PCOS patients. Although recognizing species-specific differences between mice and humans, these results suggest that, when administered for periods spanning several menstrual cycles, human treatments with DCI doses of 1200 mg/day or higher, which have a positive effect on metabolic imbalances of PCOS [65], should be carefully evaluated for their possible detrimental impact on ovarian physiology and hormonal parameters.

Intestinal absorption of inositols In addition to their detrimental effects on the ovary, high concentrations of DCI can also impair MI availability. A recent study by Garzon et al. [66] showed that 1 g DCI significantly reduces the intestinal absorption of 6 g MI. In this study, the plasma MI:DCI ratio reached 6:1, which is strongly in favor of DCI, when compared to the physiological value of 40:1 [61]. This effect can be explained by hypothesizing a competitive action on the intestinal absorption, as it emerges from the kinetics analyses of sodium-myo-inositol transporter 2 (SMIT2). This carrier protein is expressed in the small intestine and accounts for the intestinal inositol uptake, relative to either MI or DCI. In fact, SMIT2 transports MI with an average Km of 120–150 μM, with good agreement with plasma levels of MI (32.51.5 μM, ranging from 26.8 to 43.0 μM) [67]. DCI is transported with an average KM of 110–130 μM, similar to MI; nevertheless, the average plasma concentration of DCI is usually less than 100 nM, and hence, it is unlikely that it can interfere with MI absorption under normal conditions [68]. Moreover, this observation implies that in the physiological setting, SMIT2-based transport is only marginally committed in ensuring DCI absorption, and DCI could hardly impair MI uptake. However, when administered at high doses, DCI can efficiently compete with MI for intestinal absorption, thus decreasing the plasma MI:DCI ratio. Noticeably, the competitive inhibition displayed by DCI may contribute to the so-called inositol resistance—reported in some clinical trials—that could account for 30%–40% of inositol failure in treating women with PCOS [69]. In fact, when the intestinal absorption of inositol is enhanced by the co-administration of α-lactalbumin that reversibly opens enterocyte tight junctions [70], patients who were previously “resistant” to inositol recovered sensitivity, showing a significant improvement of many PCOS features [71].

Conclusion Overall, data currently available indicate that the proper MI:DCI ratio should be carefully weighted when approaching PCOS. As the therapeutic purpose focuses on how to



A clinical guide to inositols

improve ovary responsiveness to the FSH-aromatase pathway, high dosages of DCI should be avoided. After all, the fact that DCI is usually excreted in urine in large amounts, showing a low urinary MI:DCI ratio, seems to suggest that kidney can selectively concentrate and excrete DCI in excess, which can be potentially harmful [72]. However, awareness of the differences among PCOS phenotypes [73] would require—in principle—the adoption of differential treatment plans in which inositol treatment should be carefully tailored to achieve the most beneficial outcomes.

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A clinical guide to inositols

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Overcoming inositol resistance Zdravko Kamenova and Mario Montanino Olivab a

Clinic of Endocrinology, Alexandrovska University Hospital, Medical University of Sofia, Sofia, Bulgaria Altamedica Reproductive Medicine, Unicamillus Medical University, Rome, Italy


Introduction Inositols are chemically identified as hexahydroxycyclohexanes and include a family of nine stereoisomers [1]; among them, myo-inositol (MI) and D-chiro-inositol (DCI) are most widely distributed. Some studies demonstrate defects in tissue availability or altered metabolism of inositol phosphoglycans (IPGs) in PCOS patients [2,3], which are likely involved in the insulin resistance and metabolic abnormalities in these women [3]. Recently, several studies have proved their effectiveness in the treatment of metabolic and reproductive abnormalities in patients with PCOS [4], identifying them as useful and safe strategy to improve PCOS symptoms, trigger spontaneous ovulation, or induce ovulation. Despite the widely established beneficial effects of inositols on metabolic, hormonal, and reproductive PCOS clinical pictures, some women do not respond to inositol treatment, and they are classified as inositol-resistant patients. In MI-resistant patients, who failed to ovulate on monotherapy, a few different agents could be added to improve ovulation and pregnancy rate, particularly α-lactalbumin, clomiphene, and rFSH.

Effect of inositols on glucose homeostasis in PCOS MI and DCI have pivotal functions in the control of glucose homeostasis, being second messengers involved in the signaling-transduction cascade of insulin [5,6]. Both MI and DCI show insulin-mimetic properties and decrease postprandial blood glucose, while glucose metabolism is shifted toward glycogen synthesis by DCI, and toward glucose catabolism by MI [7]. Similar to metformin, MI restores the diminished GLUT-4 protein levels and glucose uptake through SMIT-1- and p-AMPK-dependent mechanism [8]. It also inhibits duodenal glucose absorption and reduces blood glucose rise [1,9], while glucose significantly counteracts cellular uptake of inositol and may induce MI depletion by the activation of the glucose-sorbitol pathway. DCI was found to be mainly involved in post-receptor insulin signaling [10,11], glycogen synthesis [12], and insulin-mediated androgen synthesis [13]. In addition, both hyperglycemia and insulin resistance modify the relative ratio in which different inositol isomers are present in these tissues. A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00009-1

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A clinical guide to inositols

A crucial effect exerted by MI and DCI in PCOS patients is the insulin-sensitizing action, which improves insulin resistance [7], reducing the hormonal, metabolic, and oxidative abnormalities in these women [14–18]. A recent study compared inositols to other treatment strategies (COC and metformin) showing that inositol therapies (either MI +folic acid or MI +DCI+folic acid) significantly improved insulin resistance and HbA1c, and reduced cholesterol and triglyceride levels and blood pressure (when used for more than 3 months). On the other hand, COC treatment worsened insulin resistance and lipid parameters, increasing cholesterol and triglyceride levels and significantly reducing FSH, LH, and SHBG serum levels concomitantly [19]. Combined treatment with MI and COC turned out to be more effective in controlling endocrine, metabolic, and clinical profiles in patients with PCOS than oral contraceptive alone. However, some beneficial effects of inositols were not observed in morbidly obese patients (BMI >37 kg/m2), and an inverse relationship between BMI and treatment efficacy was described [20], while in other studies, MI administration was more effective in obese patients with high fasting insulin plasma levels [21]. In another study, even greater effect of MI was demonstrated compared to metformin. In detail, MI supplementation significantly reduced fasting plasma glucose, serum insulin levels, HOMA-IR, serum triglycerides and VLDL-cholesterol levels, and significantly increased the quantitative insulin sensitivity check index compared to metformin [22]. On the other hand, the combination metformin +MI showed greater reduction of HOMA-IR at 3 months of treatment than metformin alone, while the effect on fasting blood glucose and insulin levels was not statistically different [23].

Effect of inositols on ovarian function in PCOS MI and DCI are abundant in follicular fluid and have a role in oocyte maturation, fertilization, implantation, and post-implantation development [24,25]. Despite the chemical similarities of MI and DCI and their synergistic effect on insulin sensitivity, they exert different functions on the ovary. In particular, MI regulates glucose uptake and FSH signaling [13,26] and is essential for proper oocyte maturation [24]. DCI is mainly responsible for insulin-mediated androgen synthesis and can act as aromatase inhibitor, while MI has an opposite effect on the activity of this enzyme [27]. Under physiological conditions, MI/DCI ratio is between 100:1 in the follicular fluid and 40:1 in plasma [28,29]. In patients with PCOS and insulin resistance, hyperinsulinemia induces higher DCI-to-MI ratio due to stimulated epimerase activity, which transforms MI to DCI. Higher MI/DCI ratios increase aromatase activity in granulosa cells, inducing estrogen biosynthesis; meanwhile, lower MI/DCI ratios stimulate androgen production in thecal cells [27]. Several studies showed that plasma LH, LH/FSH, prolactin, testosterone levels and Ferriman-Gallwey scores were significantly reduced after MI treatment [15,30,31].

Overcoming inositol resistance

Beneficial effects on hormonal profile, in terms of decreased free testosterone and LH levels and increased estradiol and SHBG, were also observed after the administration of MI +DCI in a 40:1 ratio, compared to placebo [18]. Other studies demonstrated that in patients with PCOS, MI treatment ameliorated their ovarian function and fertility [32,33]. This meant that MI became a novel method to improve spontaneous ovulation [15,20,34] or to induce ovulation [35–37]. MI turned out to be more effective in combination with metformin than metformin alone in restoring the regularity of menstrual cycle, although body weight, BMI, and waist and hip circumferences decreased significantly in all groups [38]. Compared to clomiphene citrate, MI showed a non-significant trend to lower resistance rate (30.6% vs 36.8%, P ¼0.62), lower ovulation rate (69.4% vs 79.5%, P ¼0.31), and higher pregnancy rate (33.3% vs 28.2%, P ¼0.13) [35]. MI supplementation also produced very good clinical results with a significant reduction in cancellation rate (0% vs 40%) and the consequent improvement in clinical pregnancy rate in PCOS insulin-resistant patients, undergoing gonadotropin ovulation induction with the low-dose step-down regimen [36]. The various beneficial effects of inositols on follicular development, hormonal regulation, and glucose homeostasis support their use as therapeutic agents in patients with PCOS. Many studies confirmed their positive effects both alone or in combination with other substances, enhancing their therapeutic effect and bioavailability. Moreover, it should be taken into account that MI treatment is safe and very well tolerated when compared to other therapeutic options to induce ovulation. In 2013, the International Consensus Conference on MI and DCI in Obstetrics and Gynecology recognized that both two inositols participate in several biological pathways involved in PCOS pathogenesis, and a plenty of clinical data demonstrated that inositol supplementation is beneficial for improving metabolic and reproductive aspects of this disorder [39].

Inositol resistance Despite the widely accepted beneficial effects of inositols on metabolic, hormonal, and reproductive abnormalities in PCOS, some patients do not respond to inositol treatment. The cause of this inositol resistance is not yet well understood, mostly because the trials do not assess the differences between responders and non-responders in terms of hormonal and metabolic profiles. In a study with a small cohort of women, patients who responded to MI by establishing normal ovulation frequency (n ¼6) and/or pregnancy (n ¼6) showed similar BMI, WHR, fasting insulin and glucose, OGTT response, and circulating E2 and inhibin-B concentrations, but significantly lowered testosterone levels (2.3 vs 3.4 nmol/L, respectively), higher SHBG (35.9 vs 25.8 nmol/L; P T polymorphism of MTHFR, which encodes the cytoplasmic enzyme 5,10-methylene tetrahydrofolate reductase (MTHFR), confers up to twofold increased NTD risk, with effects in both mothers and offspring [24]. While this predisposition is observed in Caucasians, it is not seen in Hispanics [25]. Other genes of folate metabolism that have been causally linked with NTDs mostly encode proteins that function in the mitochondria, where 70% of cellular 1-carbon units are produced. For example, the glycine decarboxylase (GLDC) gene is mutated in some cases of human NTDs, with loss-of-function mutations also causing NTDs in mice [26,27]. GLDC mutations are also implicated in the often fatal inborn error of metabolism, nonketotic hyperglycinemia, in which high glycine levels damage the brain development in young children. However, the NTDs that arise in mice with Gldc mutations are unrelated to glycine accumulation and result from faulty 1-C metabolism [27].

Primary prevention of NTDs—Folic acid (FA) supplementation Research in the 1970s indicated that some vitamins may be deficient in the blood of mothers with NTD-affected pregnancies [28]. Subsequently, a series of clinical trials, case-control, and other population studies showed definitively that multivitamins [29] and specifically FA [30] can lower a woman’s risk of NTD recurrence, and probably also of first NTD occurrence. In view of the demonstrated primary prevention of NTDs by FA supplements, it is often assumed that folate deficiency is the cause of NTDs. However, normal human pregnancies can be associated with reduced folate intake and availability, whereas most NTD-affected pregnancies are associated with plasma and red blood cell folate concentrations within the normal range. Quantitatively, both plasma folate and vitamin B12 levels correlate with the risk of NTD [31]. Experimental studies in mice show that folate deficiency alone is not a sufficient cause of NTDs [32], although it can enhance a genetic predisposition [33]. Hence, folate deficiency represents an important risk factor that may interact with genetic or other nongenetic influences to determine individual risks of NTD. A problem in delivering FA supplementation is the very early stage of pregnancy at which neural tube closure occurs (4th week postconception). Because folate concentrations need to be elevated by this stage to lower the risk of NTD, the recommendation is

Inositol and the prevention of adverse fetal outcomes

to take FA peri-conceptionally, beginning several weeks prior to conception and continuing until the 12th week of pregnancy. However, a large proportion of women begin taking FA supplements only later in pregnancy, while others are not aware of its benefits, and does not take FA at all [34]. Hence, voluntary supplementation is not an effective population intervention, as shown by the lack of significant decline in NTD prevalence in countries such as the United Kingdom [35], despite education campaigns to encourage FA usage, which began in the 1990s. The ineffectiveness of voluntary supplementation has led many countries, beginning with United States and Canada, to introduce mandatory fortification of staple foods with FA. For example, bread flour fortification in the United States involves addition of 140 μg FA per 100 g flour, which delivers 100–150 μg FA per day, amounting to 25%–38% of the recommended daily intake. Although this is a relatively low level of FA addition to the diet, food fortification has proven effective in reducing the NTD prevalence, both in North and South America [36,37].

Inositol and prevention of NTDs The MRC vitamin trial [30] showed a 70% reduction in NTD recurrence in pregnancies supplemented with FA, compared with those receiving nonfolate-containing supplements. Subsequently, food fortification programs have shown a 33%–59% decline in NTD prevalence, compared with historical comparison periods [37]. Hence, in all studies, an unknown proportion (perhaps 30%–60%) of NTDs persists despite the use of FA, and these NTDs may be unresponsive to FA supplementation. Reports of NTD recurrence in families despite high dose FA intake [38] further support the concept of folate nonresponsive NTDs, while mouse genetic models of NTD can be either folate-sensitive or FA-resistant [17]. This suggests that while some developmental disturbances that lead to NTDs are susceptible to correction by exogenous FA, others are not. Further prevention of NTDs, beyond that achievable using FA, requires additional measures which in future may be used alongside FA supplementation. The most promising avenue of research relates to supplementation with inositol (vitamin B8), which is an essential micronutrient for brain neural tube closure in rat embryos [39]. In the curly tail (Grhl3 gene) mouse NTD model, which is known to be FA-resistant [40], inositol was found to be effective in reducing the incidence of open spina bifida, which is the main NTD in this model [41]. Both myo- and D-chiro inositol forms were effective, and no adverse effects were detected for either mother or fetus [42]. Subsequent work showed that inositol acts to normalize neural tube closure via stimulation of a pathway involving protein kinase C activation [43] and correction of a genetically determined cellular proliferation defect in the curly tail mutant embryo. Research with inositol has now progressed into clinical studies in pregnancies at high risk of NTDs. In 2002, the first case was reported of inositol supplementation in a woman



A clinical guide to inositols

with two previous FA-resistant NTD cases [38]. Subsequently, a number of other cases of inositol therapy were described in human pregnancies at risk for putative FA-resistant NTDs [44–46]. A pilot randomized trial was undertaken to assess the feasibility of a large-scale clinical trial of inositol effectiveness for NTD prevention. The PONTI (Prevention of Neural Tube defects by Inositol) study recruited women who had experienced one or more previous NTD-affected pregnancies [47]. Among 14 pregnancies randomly assigned to periconceptional supplementation with myo-inositol (1 g) and FA (5 mg) daily, healthy unaffected babies were born in all cases. By comparison, 19 pregnancies randomly assigned to receive placebo and FA resulted in the birth of 18 normal babies, and one fetus with anencephaly. Among a further group of nonrandomized pregnancies, 21 women took inositol and FA and had no NTD recurrences, whereas 3 women took FA alone and 2 had NTD recurrences. In aggregate, therefore, inositol + FA supplementation was associated with no recurrences among 35 pregnancies whereas the FA alone (placebo) had 3 recurrences among 22 pregnancies (P ¼ .067). At the time of writing, no NTD recurrences have been observed in more than 80 women at high risk for isolated NTDs who have taken inositol and FA periconceptionally in their next pregnancies. Given that some of these women had a history of two NTD-affected pregnancies, this group of pregnancies would have been expected to exhibit three to four further recurrences. Hence, the available data are consistent with inositol having a preventive effect on NTD recurrence when taken as a supplement with FA. Regarding the safety of inositol supplementation in human pregnancy, myo-inositol has been used in many different contexts, without significant adverse effects being noted. Inositol prescription is now widespread as a treatment for polycystic ovary syndrome (PCOS) to reduce the risk of gestational diabetes and to improve oocyte quality as an adjunct to intracytoplasmic sperm injection (ICSI) and in vitro fertilization embryo transfer (IVF-ET). A metaanalysis of 965 pregnant women affected by gestational diabetes mellitus (GDM) who were randomized to receive inositol, placebo or no treatment showed no adverse events. No congenital malformations were reported in fetuses or newborns [48]. An earlier Cochrane Review of this topic similarly concluded that no adverse events have been associated with inositol antenatal supplementation [49]. Both myo-inositol (1.75 g) and D-chiro-inositol (250 mg) were used in the treatment of 68 women until 24th week of the pregnancy, with no adverse effects reported for either mother or the fetus [50]. Hence, even at a dosage in excess of the typical 0.5–1 g daily, myo-inositol has proven safe for use in pregnant women.

Counseling families at risk of NTD recurrence When counseling a couple about their recurrence risk after a pregnancy affected by NTD, a structured approach is needed. This includes ascertainment of the precise

Inositol and the prevention of adverse fetal outcomes

diagnosis for the previous affected pregnancy (or multiple pregnancies), determination of the context in which the previous NTD occurred (e.g., FA usage), and identification of any etiological or predisposing factors that might affect future recurrence risk. Parents typically ask: “what happened?,” “why did it happen?,” and “will it happen again in our family?.” The counselor’s job is to offer the most accurate possible answers to these questions, in a form that is fully comprehensible to the parents. In our experience, families are often told by a medical or nursing professional that their NTD-affected pregnancy is “just a chance occurrence which is unlikely to happen again.” They may be advised to take high dose (5 mg) FA when planning another pregnancy and to “hope for the best.” Having received often less than helpful responses from medical professionals, many parents access web sites for additional information, and this can give them conflicting facts and opinions and lead to confusion and uncertainty on how to prepare for a future pregnancy. Families regularly approach the authors at this stage, often saying this was their “last resort,” having consistently failed to obtain satisfactory information from other sources. We suggest that the following information should be reviewed, wherever possible, when preparing to counsel a family with a previous NTD-affected pregnancy. To establish a diagnosis, a fetal ultrasound evaluation of the putative NTD together with report of any associated abnormalities is essential, together with pre- and postnatal MRIs wherever available. Clinical, genetic, and/or pathological evaluations are valuable, but fetal postmortem examination may not be undertaken as NTDs are considered relatively “common” and follow-up investigations are often deemed unnecessary. To establish predisposing factors in the family, a detailed medical history is needed from each parent, including pregnancy and family histories, and exposure to drugs particularly anticonvulsants. Any previous occurrence of birth defects, fetal losses, and stillbirths needs to be documented, a family pedigree of at least three generations should be drawn, and potential for consanguinity assessed. Cytogenetic and/or array comparative genomic hybridization (aCGH) analysis of the fetus, newborn, and/or parents may be valuable, although most “sporadic” NTDs yield no abnormalities on these examinations. Maternal blood homocysteine, serum and red blood cell folate, and vitamin B12 levels can be obtained, and MTHFR genotype may be considered, although this should not be used to predict recurrence risk for individual cases. With this information at hand, it may occasionally be possible to indicate a specific cause for the family’s NTD, although in most cases such likely causation will not be evident. Then, the counselor will indicate the relevant risk of NTD recurrence and enquire in particular about previous FA usage. For NTDs, empirical risks are used to inform counseling for recurrence rates, as causation is unknown in the great majority of cases. After 1 NTD-affected pregnancy, the recurrence risk is usually estimated at around 3%–5% (i.e., 1 in 20–30), and this rises to around 10%–20% (1 in 5–10) after 2 affected pregnancies. First degree relatives are usually counseled as having similar recurrence rates



A clinical guide to inositols

as women who have experienced an NTD pregnancy themselves, whereas lower recurrence risk are quoted as the closeness of relationship to the index case declines. Women often say they took 400 μg FA daily in the previous pregnancy, but the frequency and period of pregnancy when supplementation occurred must be established. Most important is whether FA usage was peri-conceptional (i.e., began before conception) or only following confirmation of the pregnancy. In the latter case, the NTD likely arose in the absence of FA supplementation. For women who did not take FA periconceptionally, the most important recommendation is to take high dose (5 mg) FA in preparation for the next pregnancy. For women who give a convincing report of periconceptional FA usage in the NTDaffected pregnancy, it is appropriate to discuss the concept of folate-nonresponsiveness, with a lay summary of the evidence being given. This is often the first time that a family has met with the concept that FA may not be able to prevent all NTDs. They often ask if a test is available to show whether they are FA responsive or nonresponsive, and should be told that currently no test is available, as the underlying mechanism(s) of FA responsiveness is unknown. High-dose FA should be recommended, and in addition, the parents can be told about the possibility that inositol supplementation may be beneficial, particularly for FA-nonresponsive cases. It is important to stress that inositol usage is still in the research stage and that its efficacy is not yet proven in a large-scale clinical trial. The safety of inositol should also be addressed, with a statement that it has so far proven safe in pregnancy, but that adverse effects could emerge with more widespread usage. Parents should be asked to check with the clinician supervising their pregnancies, before embarking on inositol supplementation. If a family is interested in using inositol, the timing (per-conceptional, until the 12th week of pregnancy), dose (1 g per day) and formulation (pure myo-inositol without additional active ingredients) should be described. Sources of myo-inositol (0.5 g tablets or capsules) should be indicated, so that parents can buy directly from a commercial supplier. Follow-up is extremely valuable for increasing the evidence base, and all families should be asked to provide feedback on future pregnancies, with details of their chosen supplementation regime, wherever relevant.

Conclusions and future directions Primary prevention of NTDs by FA is a clinical reality but in practice this is incomplete, with as many as 60% of cases proving nonresponsive to FA. Inositol has an expanding evidence base suggesting effectiveness in prevention of FA-nonresponsive cases and is currently being used by families on a case-by-case basis. A future large-scale clinical trial is needed to establish whether inositol, in combination with FA, is truly effective in preventing a higher proportion of NTDs than FA alone. It remains to be established whether inositol supplementation should also be recommended for low risk pregnancies

Inositol and the prevention of adverse fetal outcomes

(i.e., those without a family history of NTD). Historically, the effectiveness of FA supplementation was first demonstrated for NTD recurrence, and then subsequent studies supported prevention of first occurrence NTDs as well. It seems reasonable to follow a similar route toward population-wide usage of inositol.

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[19] Galea GL, Nychyk O, Mole MA, Moulding D, Savery D, Nikolopoulou E, Henderson DJ, Greene NDE, Copp AJ. Vangl2 disruption alters the biomechanics of late spinal neurulation leading to spina bifida in mouse embryos. Dis Model Mech 2018;11:dmm032219. [20] Ybot-Gonzalez P, Savery D, Gerrelli D, Signore M, Mitchell CE, Faux CH, Greene NDE, Copp AJ. Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure. Development 2007;134:789–99. [21] Chen Z, Lei Y, Zheng Y, Aguiar-Pulido V, Ross ME, Peng R, Jin L, Zhang T, Finnell RH, Wang H. Threshold for neural tube defect risk by accumulated singleton loss-of-function variants. Cell Res 2018;28:1039–41. [22] Brouns MR, de Castro SC, Terwindt-Rouwenhorst EA, Massa V, Hekking JW, HIrst CS, Savery D, Munts C, Partridge D, Lamers W, Kohler E, van Straaten HW, Copp AJ, Greene ND. Overexpression of Grhl2 causes spina bifida in the axial defects mutant mouse. Hum Mol Genet 2011;20:1536–46. [23] Juriloff DM, Harris MJ. A consideration of the evidence that genetic defects in planar cell polarity contribute to the etiology of human neural tube defects. Birth Defects Res A Clin Mol Teratol 2012;94:824–40. [24] Van der Put NMJ, Eskes TKAB, Blom HJ. Is the common 677C–>T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects? A meta-analysis. Q J Med 1997;90:111–5. [25] Amorim MR, Lima MA, Castilla EE, Orioli IM. Non-Latin European descent could be a requirement for association of NTDs and MTHFR variant 677C > T: a meta-analysis. Am J Med Genet A 2007;143A:1726–32. [26] Narisawa A, Komatsuzaki S, Kikuchi A, Niihori T, Aoki Y, Fujiwara K, Tanemura M, Hata A, Suzuki Y, Relton CL, Grinham J, Leung KY, Partridge D, Robinson A, Stone V, Gustavsson P, Stanier P, Copp AJ, Greene ND, Tominaga T, Matsubara Y, Kure S. Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans. Hum Mol Genet 2012;21:1496–503. [27] Pai YJ, Leung KY, Savery D, Hutchin T, Prunty H, Heales S, Brosnan ME, Brosnan JT, Copp AJ, Greene ND. Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice. Nat Commun 2015;6:6388. [28] Smithells RW, Sheppard S, Schorah CJ. Vitamin deficiencies and neural tube defects. Arch Dis Child 1976;51:944–50. [29] Smithells RW, Nevin NC, Seller MJ, Sheppard S, Harris R, Read AP, Fielding DW, Walker S, Schorah CJ, Wild J. Further experience of vitamin supplementation for prevention of neural tube defect recurrences. Lancet 1983;1:1027–31. [30] MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1991;338:131–7. [31] Kirke PN, Molloy AM, Daly LE, Burke H, Weir DG, Scott JM. Maternal plasma folate and vitamin B12 are independent risk factors for neural tube defects. Q J Med 1993;86:703–8. [32] Heid MK, Bills ND, Hinrichs SH, Clifford AJ. Folate deficiency alone does not produce neural tube defects in mice. J Nutr 1992;122:888–94. [33] Burren KA, Savery D, Massa V, Kok RM, Scott JM, Blom HJ, Copp AJ, Greene NDE. Geneenvironment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function. Hum Mol Genet 2008;17:3675–85. [34] McNulty B, Pentieva K, Marshall B, Ward M, Molloy AM, Scott JM, McNulty H. Women’s compliance with current folic acid recommendations and achievement of optimal vitamin status for preventing neural tube defects. Hum Reprod 2011;26:1530–6. [35] Abramsky L, Botting B, Chapple J, Stone D. Has advice on periconceptional folate supplementation reduced neural-tube defects? Lancet 1999;354:998–9. [36] Berry RJ, Bailey L, Mulinare J, Bower C. Fortification of flour with folic acid. Food Nutr Bull 2010;31: S22–35. [37] Rosenthal J, Casas J, Taren D, Alverson CJ, Flores A, Frias J. Neural tube defects in Latin America and the impact of fortification: a literature review. Public Health Nutr 2014;17:537–50.

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[38] Cavalli P, Copp AJ. Inositol and folate-resistant neural tube defects. J Med Genet 2002;39:e5. [39] Cockroft DL. Changes with gestational age in the nutritional requirements of postimplantation rat embryos in culture. Teratology 1988;38:281–90. [40] Seller MJ. Vitamins, folic acid and the cause and prevention of neural tube defects. In: Bock G, Marsh J, editors. Neural tube defects (Ciba foundation symposium 181). Chichester: John Wiley & Sons; 1994. p. 161–73. [41] Greene NDE, Copp AJ. Inositol prevents folate-resistant neural tube defects in the mouse. Nat Med 1997;3:60–6. [42] Cogram P, Tesh S, Tesh J, Wade A, Allan G, Greene NDE, Copp AJ. D-chiro-inositol is more effective than myo-inositol in preventing folate-resistant mouse neural tube defects. Hum Reprod 2002;17:2451–8. [43] Cogram P, Hynes A, Dunlevy LPE, Greene NDE, Copp AJ. Specific isoforms of protein kinase C are essential for prevention of folate-resistant neural tube defects by inositol. Hum Mol Genet 2004;13:7–14. [44] Cavalli P, Tedoldi S, Riboli B. Inositol supplementation in pregnancies at risk of apparently folateresistant NTDs. Birth Defects Res A Clin Mol Teratol 2008;82:540–2. [45] Cavalli P, Tonni G, Grosso E, Poggiani C. Effects of inositol supplementation in a cohort of mothers at risk of producing an NTD pregnancy. Birth Defects Res A Clin Mol Teratol 2011;91:962–5. [46] Cavalli P, Ronda E. Myoinositol: the bridge (PONTI) to reach a healthy pregnancy. Int J Endocrinol 2017;2017:5846286. [47] Greene NDE, Leung KY, Gay V, Burren KA, Mills K, Chitty LS, Copp AJ. Inositol for prevention of neural tube defects: a pilot randomised controlled trial. Br J Nutr 2016;115:974–83. [48] Vitagliano A, Saccone G, Cosmi E, Visentin S, Dessole F, Ambrosini G, Berghella V. Inositol for the prevention of gestational diabetes: a systematic review and meta-analysis of randomized controlled trials. Arch Gynecol Obstet 2019;299:55–68. [49] Crawford TJ, Crowther CA, Alsweiler J, Brown J. Antenatal dietary supplementation with myoinositol in women during pregnancy for preventing gestational diabetes. Cochrane Database Syst Rev 2015;12:CD011507. [50] Dell’Edera D, Sarlo F, Allegretti A, Epifania AA, Simone F, Lupo MG, Benedetto M, D’Apice MR, Capalbo A. Prevention of neural tube defects and maternal gestational diabetes through the inositol supplementation: preliminary results. Eur Rev Med Pharmacol Sci 2017;21:3305–11.



Inositol supplementation for preventing gestational diabetes mellitus Fabio Facchinettia, Rosario D’Annab, and Moshe Hodc,d a

UOC Gynecology and Obstetrics, Mother-Infant Department, University of Modena, Modena, Italy Department of Human Pathology, University of Messina, Messina, Italy c Mor Comprehensive Women’s Health Care Center, Tel-Aviv, Israel d FIGO, Pregnancy and Non-Communicable Diseases Committee, London, United Kingdom b

Gestational diabetes mellitus (GDM) around the World: Basics and problematics Originally, the relevance of diagnosing gestational hyperglycemia was linked to an increased risk of future type 2 diabetes mellitus (T2DM) in the mother. In recent years, considerable progress has been made in understanding the maternal metabolic adaptations induced by the feto-placental unit during pregnancy and the impact of its derangement on fetal development and pregnancy outcomes. Consequently, clinicians have become aware of the need to properly identify and manage metabolic dysregulation in pregnancy, in particular, aberrations in glucose metabolism. This has led to increased focus on the ability to predict and prevent many potential fetal and maternal complications due to hyperglycemia in pregnancy (HIP) [1].

Classification and definition The classification of HIP and the definition of gestational diabetes mellitus (GDM) are evolving. Until recently, the accepted definition of GDM was “any degree of glucose intolerance with onset or first recognition during pregnancy” [2]. This definition includes women with preexisting diabetes who may not have been identified prior to pregnancy and blurs the line between morbidities associated with diabetes in pregnancy and gestational diabetes. Renewed efforts are being made to improve the definition and classification of hyperglycemia during pregnancy. These efforts are also spurred by the increasing prevalence of diabetes and GDM [3] and greater risk of maternal and fetal complications resulting from diabetes mellitus antedating pregnancy. Therefore, hyperglycemia first detected at any time during pregnancy should be classified either as diabetes mellitus in pregnancy or GDM [4].

A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00011-X

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A clinical guide to inositols

Diabetes in pregnancy Diabetes in pregnancy may be either preexisting diabetes (type 1 or type 2) antedating pregnancy, or overt diabetes first diagnosed during pregnancy. Apart from the severity, the fact that hyperglycemia may be present at conception and during embryogenesis it increases the vulnerability to disrupted organogenesis, resulting in higher chances of spontaneous abortions and congenital malformations. Diabetes in pregnancy may also result in greater macrosomia and higher risk of obstructed labor, shoulder dystocia, and neonatal hypoglycemia. In addition, vascular complications such as retinopathy or nephropathy may be first detected or get worsened during pregnancy. When hyperglycemia was first detected on routine testing anytime during the course of pregnancy in women without the history of known diabetes meets the criteria for diagnosis of diabetes in the nonpregnant state (Fasting Plasma Glucose (FPG) 7.0 mmol/L or 126 mg/dL and/or 2-h 75 g OGTT value11.0 mmol/L or 200 mg/dL or Random Plasma Glucose (RPG)11.0 mmol/L or 200 mg/dL associated with signs and symptoms of diabetes): the condition is called diabetes in pregnancy. The vulnerability to complications is high because of the degree of hyperglycemia and the uncertainty as to when the hyperglycemia started; was it present prior to pregnancy or during early pregnancy? While diabetes diagnosed first time in pregnancy maybe type 1 or type 2, a diagnosis of type 2 is more likely. Compared to gestational diabetes, diabetes in pregnancy is likely to manifest and be detected earlier, as early as the first trimester.

Gestational diabetes mellitus (GDM) In 2014, the International Federation of Gynecology and Obstetrics (FIGO) embarked on a new GDM initiative with the ambitious objectives of (1) raising awareness of the links between hyperglycemia and poor maternal and fetal outcomes as well as to the future health risks to mother and offspring and demanding a clearly defined global health agenda to tackle this issue; and (2) creating a consensus document that provides guidance for testing, management, and care of women with GDM regardless of resource setting and disseminating and encouraging its use. To develop such international guidance, FIGO brought together a group of experts to develop a document to frame the issues around gestational diabetes and suggest key actions to address the health burden posed by it [5]. The following document was created “The International Federation of Gynecology and Obstetrics (FIGO) Initiative on gestational diabetes mellitus: A pragmatic guide for diagnosis, management, and care” which was published in the International Journal of Gynecology and Obstetrics and launched at FIGO World Congress in October 2015 in Vancouver. Hyperglycemia is one of the most common medical conditions women encounter during pregnancy with an estimated one in six live births (16.8%) is to women with some

Inositol supplementation for preventing gestational diabetes mellitus

form of hyperglycemia in pregnancy. While 16% of these cases may be due to diabetes in pregnancy (either preexisting diabetes—type 1 or type 2—which antedates pregnancy or is first identified during testing in the index pregnancy), the majority (84%) is due to GDM. The occurrence of GDM parallels the prevalence of impaired glucose tolerance (IGT), obesity, and T2DM in a given population. These conditions are on the rise globally. Moreover, the age of onset of diabetes and prediabetes is declining while the age of child-bearing is increasing. There is also an increase in the rate of overweight and obese women of reproductive age; thus, more women entering pregnancy have risk factors that make them vulnerable to hyperglycemia during pregnancy. GDM is associated with a higher incidence of maternal morbidity including cesarean deliveries, shoulder dystocia, birth trauma, hypertensive disorders of pregnancy (including preeclampsia), and subsequent development of T2DM. Perinatal and neonatal morbidities also increase; the latter includes macrosomia, birth injury, hypoglycemia, polycythemia, and hyperbilirubinemia. Long-term sequelae in offspring with in utero exposure to maternal hyperglycemia may include higher risks for obesity and diabetes later in life. In most parts of low- and middle-income countries (LMICs) (which contribute to over 85% of the annual global deliveries), the majority of women are either not screened or improperly screened for diabetes during pregnancy—despite that these countries account for 80% of the global diabetes burden as well as 90% of all cases of maternal and perinatal deaths and poor pregnancy outcomes. In particular, eight LMICs—India, China, Nigeria, Pakistan, Indonesia, Bangladesh, Brazil, and Mexico—account for 55% of the global live births (70 million live births annually) as well as 55% of the global burden of diabetes (209.5 million) and should be key targets for any focused strategy on addressing the global burden of GDM pregnancies. These countries have been identified as priority countries for all future GDM interventions. Given the interaction between hyperglycemia and poor pregnancy outcomes, the role of in utero imprinting in increasing the risk of diabetes and cardiometabolic disorders in the offspring of mothers with hyperglycemia in pregnancy, as well as increasing maternal vulnerability to future diabetes and cardiovascular disorders, there needs to be a greater global focus on preventing, screening, diagnosing, and managing hyperglycemia in pregnancy. The relevance of GDM as a priority for maternal health and its impact on the future burden of noncommunicable diseases is no longer in doubt; but how best to deal with the issue remains contentious as there are many gaps in knowledge on how to prevent, diagnose, and manage GDM to optimize care and outcomes. These must be addressed through future research. Fetal implications: Growth and development of the human conceptus occur within the metabolic milieu provided by the mother. As early as 1954, Pedersen et al. [6] demonstrated that newborns of diabetic mothers suffered from hypoglycemia and hypothesized



A clinical guide to inositols

that this was due to fetal hyperinsulinism as a consequence of increased transplacental transfer of sugar. Van Assche and Gepts [7] confirmed hyperplasia of the insulinproducing beta cells in infants of diabetic mothers and postulated that the hyperplasia was related to beta-cell hyperactivity which could have consequences in later life. In animal experiments, Aerts and Van Assche [8] showed that modifications in the endocrine pancreas during intrauterine life caused persistent changes that manifest in later adult life (in the second generation). Though not perceptible under basal conditions, these changes become apparent in situations stressing the beta-cell activity, such as pregnancy. Pregnancy in second-generation rats showed increased nonfasting blood glucose, with no apparent adaptation of the beta cells. This inadequate adaptation to pregnancy caused changes in the fetal endocrine pancreas in fetuses of the third generation, thereby suggesting a transgenerational transmission of risk. It is now evident that an abnormal intrauterine environment has consequences in later life mediated through epigenetic changes. This phenomenon is known as developmental programming. An increasing body of evidence supports the hypothesis that the abnormal metabolic environment of the mother with diabetes mellitus affects the developing fetal tissues, organs, and control mechanisms, eventually leading to long-term structural changes and functional implications in adult life. The fetal tissues most likely to be affected are neural cells, adipocytes, muscle cells, and pancreatic beta cells. Freinkel [9] introduced the concept of pregnancy as a “tissue culture experiment,” in which the placenta and the fetus develop in an “incubating medium” totally derived from maternal fuels. All these fuels traverse the placenta from the maternal compartment either with (e.g., glucose, lipids) or against (e.g., amino acids) concentration gradient and create to the fetal milieu. Since these constituents are regulated in part by maternal insulin, disturbances in its supply or action influence the growth medium to which the fetus is exposed. Maternal hyperglycemia leads to fetal hyperglycemia and eventually to fetal hyperinsulinemia. According to Freinkel’s hypothesis, the abnormal mixture of metabolites from the mother modifies the phenotypic expression of newly formed fetal cells, which in turn determine permanent, short-, and long-term effects in the offspring. Depending upon the timing of (embryonic-fetal) exposure to the aberrant fuel mixture, different events may develop. Early in the first trimester, intrauterine growth restriction and organ malformation, described by Freinkel as “fuel-mediated teratogenesis,” may occur. During the second trimester, at the time of brain development and differentiation, behavioral, intellectual, or psychological damage may occur. During the third trimester, abnormal proliferation of fetal adipocytes and muscle cells, together with hyperplasia of pancreatic beta cells and neuroendocrine cells, may be responsible for the development of obesity, hypertension, and T2DM later in life. Maternal implications: Until the discovery of insulin by Banting and Best in 1921, very few women with diabetes became pregnant spontaneously, and even fewer achieved a successful pregnancy outcome. At that time, about 50% of women died during pregnancy from diabetes-related complications (mainly ketoacidosis) and about 50% of the fetuses

Inositol supplementation for preventing gestational diabetes mellitus

failed to develop in utero. The situation has improved remarkably in the last two to three decades in the developed world but even now women with diabetes mellitus have a markedly higher risk for a number of poor pregnancy outcomes described earlier. These complications, together with the increased rate of vascular dysfunction (retinopathy and nephropathy), contribute to higher maternal morbidity and mortality among patients with diabetes mellitus. Also, hyperglycemia first appearing during pregnancy is associated with a high risk of developing diabetes and cardiovascular diseases in later life [10–13]. Public health implications: In most parts of the low, low middle, and upper middleincome countries which contribute to over 85% of the annual global deliveries and over 90% of all cases of maternal and perinatal deaths and poor pregnancy outcomes as well as 80% of the global diabetes burden—more than half of them undiagnosed; the majority of women are not properly screened for diabetes during pregnancy. Given the interaction between hyperglycemia and maternal and perinatal morbidity and mortality and the role of in utero imprinting in increasing risk of diabetes and cardio-metabolic disorders in offspring of mothers with hyperglycemia in pregnancy as well as increasing maternal vulnerability to future diabetes and cardiovascular disorders, there needs to be a greater focus on preventing, screening, diagnosing, and managing hyperglycemia in pregnancy globally. There is adequate evidence as described later that appropriate lifestyle changes continued after delivery help prevent or delay the onset of T2DM in women with GDM. The relevance of GDM as a priority for maternal health and its impact on the future burden of noncommunicable diseases is no longer in doubt but how best to deal with the issue remains relatively unclear as health systems are not integrated and work in silos of maternal, newborn, and child health (MNCH); noncommunicable diseases (NCDs); health promotion and disease prevention; and clinical care delivery with little or no interaction among them. The importance of pregnancy as an eminent “teachable” moment to institute healthy lifestyle practices for the whole family is also missed. There are also many gaps in knowledge on how best to prevent, diagnose, and manage GDM to optimize care and outcomes.

Diagnosing GDM Problems of multiple criteria: There are a plethora of diverse algorithms for screening and diagnosis of GDM. Tragically, even the endocrine, diabetes, and obstetric associations within the same country recommend different protocols and cut-off values. These recommendations have been criticized for lacking validation as they were either (a) developed based on tenuous data or (b) result of expert-opinions or (c) biased due to economic considerations or (d) convenience-oriented [14] and create confusion and uncertainty amongst care givers. The problem, as shown consistently by several studies including the hyperglycemia and adverse pregnancy outcomes (HAPO) study, is that the risk of hyperglycemia-associated poor outcomes is continuous with no clear infliction



A clinical guide to inositols

points [15–18]. It is therefore clear that any set of criteria proposed will need to evolve from a consensus approach, balancing risks and benefits, in particular, social, economic, and clinical contexts [19]. Cut-off values for fasting, 1-hr, and 2-h 75 g OGTT test based on an acceptable odds ratio for markers of diabetic fetopathy (large for gestational age (LGA), excess fetal adiposity, and fetal hyperinsulinemia) in the HAPO study were proposed by the International Association of Diabetes in Pregnancy Study Groups (IADPSG) [20]. These cut-off values were accepted and endorsed by the WHO [4]. However, LGA and fetal adiposity are not solely dependent maternal glucose alone. For example, in using a 2-h glucose cut-off value of 8.5 mmol/L or 153 mg/dL based on an odds ratio of 1.75 for adverse outcomes derived from HAPO data as per IADPSG recommendation may not be as efficient in identifying women at risk for fetal overgrowth as those identified by having a 2-h glucose corresponding to that at a slightly lower odds ratio, e.g., 1.5. The latter corresponds to the older WHO criteria 2-h value of 7.8 mmol/L or 140 mg/dL. This may be particularly relevant in the developing countries particularly in south Asia where women are relatively small, and a larger baby may pose greater obstetric risk. Apart from the different cut-off values, the lack of consensus among the different professional bodies on the algorithm for screening and diagnosis of GDM is perhaps an even bigger problem. Despite repeated pleas for a single process and criteria [21], the ideal protocol for the diagnosis of GDM continues to be debated. Universal versus selective testing: Selective testing based on clinical risk factors for GDM evolved from the view that in populations with low risk of GDM subjecting all pregnant women to a laboratory test was considered a waste of resources. Some of the risk factors used are age and BMI (varying thresholds), race, polyhydramnios, macrosomia (present or past pregnancy), GDM in past, unexplained stillbirth, T2DM in first-degree relative, and polycystic ovary syndrome (PCOS). The tri-hospital gestational diabetes project [22] developed a scoring system based on maternal age, BMI, and race. However, variations in risk factors have resulted in different and generally poor sensitivity and specificity. The major problem of risk-factor based screening is that it is quite demanding for the healthcare provider; also, the more complex the protocol for testing, the lower likelihood for compliance by both clients and care providers. This applies particularly when ascertainment of risk factors is likely to be poor due to low education and awareness and poor records. Moreover, many of the low resource countries also have ethnic populations considered at high risk and so there seems little justification for selective testing. It has been elegantly argued that risk factor or glucose challenge test (GCT)-based screening to identify women who should be tested has no place in the diagnosis of GDM [23]. Screening on the basis of risk factors will require most women to be tested and will inevitably and unknowingly miss women with GDM. GCT screening misses many of those with a modestly elevated fasting glucose and runs the risk of missing other women with GDM because of the inevitable high no-show rate following a positive screen. It is open to speculation how the combination of risk factor screening and a GCT may compound the number of missed diagnoses. It has been proposed that in

Inositol supplementation for preventing gestational diabetes mellitus

the overall cost of delivering care to women with GDM, the cost of administering a glucose tolerance test to all pregnant women is likely to be minor if the initial fasting GTT level result can be used to decide if the full GTT is needed [24]. In situations where women may not attend antenatal clinics fasting and may not be able to come for testing while fasting for the second step test of the GCT, a single-step 75 g 2-h nonfasting test, as is used in India, may be applied [25]. Implementation of guidelines is a constant challenge. The reality is that most lowresource countries around the world are unable to implement a GDM detection program based on a universal 75 g OGTT or rely on just high-risk women undergoing a 75g OGTT. These challenges and barriers have been reviewed extensively [26]. The applicability of the IADPSG cut-off value for fasting glucose to diagnose GDM, especially in the first trimester, has been contested in a recent study from China [27]. The FIGO guideline [28] provides a practical and pragmatic guide for national associations to adopt and promote a uniform approach to testing, diagnosis, and management of GDM for all countries and regions based on their financial, manpower, and infrastructure resources (Table 1).

Diagnostic criteria Diabetes in pregnancy: The diagnosis of diabetes in pregnancy should be made by one or more of the following results recorded anytime on routine testing during the course of pregnancy: (1) Fasting plasma glucose 7.0 mmol/L (126 mg/dL) (2) 2-h plasma glucose 11.1 mmol/L (200 mg/dL) following a 75-g oral glucose load Table 1 Options for diagnosis of GDM based on resource settings. Strategy Setting

Who to test and when

Fully resourced settings

All women at booking/first trimester 24–28 weeks

Fully resourced settings serving ethnic populations at high risk

All women at booking/first trimester 24–28 weeks

Any setting (basic); particularly medium- to low-resource settings serving ethnic populations at risk

All women between 24 and 28 weeks

Diagnostic test


Measure FPG, RBG, or HbA1c to detect diabetes in pregnancy If negative: perform 75 g 2-h OGTT Perform 75 g 2-h OGTT to detect diabetes in pregnancy If negative: repeat 75 g 2-h OGTT Perform 75 g 2-h OGTT

1 jO

2 jOOO

1 jO



A clinical guide to inositols

(3) Random plasma glucose 11.1 mmol/L (200 mg/dL) in the presence of diabetes symptoms (4) HbA1C6.5% GDM: As per the recommendation of the IADPSG (2010) and WHO (2013), the diagnosis of GDM is made using a single step 75 g OGTT when one or more of the following results are recorded on routine testing between 24 and 28 weeks of pregnancy or at any other time during the course of pregnancy: (1) Fasting plasma glucose 5.1–6.9 mmol/L (92–125 mg/dL) (2) 1-h post 75 g oral glucose load 10 mmol/L (180 mg/dL) (3) 2-h post 75 g oral glucose load 8.5–11.0 (mmol/L) (153–199 mg/dL) Management of HIP: Pregnancies complicated by hyperglycemia maternal and fetal outcomes are directly correlated with maternal glycemic control, and therefore, the primary goal must be to ensure maternal glucose levels as close to normal as possible throughout pregnancy. Antenatal follow-up: There is no high-quality evidence, or for that matter, any evidence to support a particular protocol of antenatal follow-up for women with diabetes [28]. Fetal sonographic assessment: Monitoring fetal growth is both challenging and inaccurate, with a 15% error margin. Since fetal macrosomia is the most frequent complication of diabetes, a particular effort should be directed toward its diagnosis and prevention. Fetal well-being: Fetal assessment can be achieved by a fetal kick count, biophysical profile, and cardiotocography (nonstress test). There is no high-quality evidence to support a particular follow-up protocol. However, it is assumed that with reassuring fetal well-being, pregnancy prolongation to term can be achieved [28]. Timing and mode of delivery: Maternal hyperglycemia and macrosomia are associated with increased risk of intrauterine fetal death and other adverse outcomes. Therefore, induction of labor may be considered at 38–39 weeks, although there is no good-quality evidence to support such an approach. Thus, some guidelines suggest that a pregnancy with good glycemic control and a seemingly appropriate estimated weight for gestational age fetus ought to continue until 40–41 weeks [29–31]. Given the significantly greater risk of shoulder dystocia at any birthweight above 3750 g for babies of women with diabetes, consideration may be given to elective cesarean delivery when the best estimate of fetal weight exceeds 4000 g [32]. To deal with the issue of HIP, the FIGO recommends the following: • There should be greater attention and focus on the links between maternal health and NCDs and efforts made for greater integration of services. • All pregnant women should be tested for hyperglycemia during pregnancy. FIGO encourages its member associations to adapt and promote strategies to ensure universal testing of all pregnant women for hyperglycemia during pregnancy.

Inositol supplementation for preventing gestational diabetes mellitus

• WHO (2013) criteria for diagnosis of overt diabetes mellitus in pregnancy, and the WHO (2013) and IADPSG (2010) criteria for diagnosis of GDM must be used whenever possible but keeping in mind the resource constraints in many parts of the developing world alternate strategies described in the chapter should also be considered equally acceptable. • Diagnosis of GDM should be based on venous plasma samples properly collected and transported and tested in a lab. However, in primary care settings particularly in the developing world where proper facilities to test or store and transport blood samples to a distant lab may not exist, a plasma-calibrated handheld glucometer with properly stored test strips to measure plasma glucose is an acceptable alternate. Using a glucose meter in this situation may be more reliable than lab tests done on samples that have been inadequately handled and transported. • Nutrition counselling and physical activity are the primary tools in the management of GDM. Women with GDM must receive practical nutrition education and counselling that empowers them to choose the right quantity and quality of food and level of physical activity. Women with GDM must be repeatedly advised and encouraged to continue the same healthy lifestyle even after delivery in order to reduce the risk of future obesity, T2DM, and cardiovascular diseases. • When lifestyle changes are inadequate in controlling hyperglycemia, insulin may be added. Glyburide and/or metformin may be used as a safe and effective treatment option for GDM during the 2nd and 3rd trimesters in place of insulin as long as glycemic control is achieved. These medications may be initiated as a first line treatment when lifestyle modification alone fails to achieve glucose control. Insulin should be added if lifestyle and oral agents fail to control glucose levels. • Following a GDM pregnancy, the postpartum period provides an important platform to initiate early preventive health for both the mother and the child as both are at heightened risk for future obesity, metabolic syndrome, diabetes, hypertension, and cardiovascular disorders. Obstetricians must establish links with family physicians, internists, and pediatricians to support postpartum follow-up of GDM mothers linked to their children’s vaccination program to ensure continued follow-up and engagement of the high-risk mother child pair. • Public health measures to increase awareness and acceptance of preconception counselling and to increase affordability and access to preconception and antenatal services for women in the reproductive age must be put in place, as this likely to have both immediate and lasting benefit for maternal and child health.

GDM-related complications GDM is defined as any degree of glucose intolerance that occurs for the first time or is first detected during pregnancy but does not fulfill the criteria of overt diabetes [33].



A clinical guide to inositols

Prevalence of GDM depends on screening criteria, which are different also in Western countries, but the related complications are well known and common to all countries. In particular, pregnant women with GDM have an increased risk of hypertensive disorders, preterm birth, and cesarean section; the fetus experiences macrosomia frequently, rarely malformations and stillbirth; the neonate may suffer from hypoglycemia, jaundice, respiratory distress syndrome (RDS), and birth trauma as consequences of shoulder dystocia. Long-term consequences are those in childhood with an increased rate of obesity, metabolic syndrome, and sometimes T2DM; and finally, a high risk for the mother to develop T2DM later in the life.

Maternal complications Hypertensive syndromes may increase the risk of mortality and morbidity among women with GDM. In a large study, involving thousands of pregnant women affected by gestational hypertension, mild or severe preeclampsia, and eclampsia, a statistically significant correlation was found between GDM with all the hypertensive syndromes features [34]. The authors showed that women with GDM had a 50% higher risk to develop hypertensive syndromes compared to nondiabetic women [34]. This association was stronger in women who received less prenatal care. This data are in accordance with another study in which preeclampsia correlated significantly with the severity of GDM, highlighting the importance of adequate prenatal care [35]. In this last study, there were more obese women in the preeclamptic group than in the control group and the abundant fat tissue might have a nonsecondary role in determining gestational hypertension [35]. There are some shareable theories on the role of insulin resistance in inducing gestational hypertension. It is well known that insulin resistance may activate the sympathetic nervous system and an increased expression of receptors for endothelin, which is a strong vasoconstrictor [35]. Furthermore, an impaired maternal glucose level with persistent hyperglycemia may attenuate endothelium-dependent vasodilation with less nitric oxide production [36]. All these actions lead to endothelial dysfunction with less prostacyclin production and consequently persistent vasospasm and hypertension.

Fetal and neonatal complications Among fetal complications of GDM pregnancies, macrosomia defined as a birth weight 4000 g or 4500 g (depending by the size and ethnicity of pregnant women) is the most common, with a prevalence that may reach 45% among newborns from diabetic mothers [37]. The pathophysiology of macrosomia is commonly explained based on extended Pedersen’s hypothesis [38] about impaired maternal glycemic control which lead to a high amount of glucose that crosses the placenta. In particular, fetal birth weight correlates with second- and third-trimester postprandial blood sugar levels and not with fasting glucose levels [39]. From the second trimester, fetus begins to secrete insulin, which is an

Inositol supplementation for preventing gestational diabetes mellitus

anabolic hormone determining central deposition of subcutaneous fat in the abdominal and interscapular areas [40]. Consequently, a decreased head-to-shoulder ratio may cause shoulder dystocia that in few cases may evolve in brachial plexus trauma and Erb’s palsy. About risk factors for shoulder dystocia, GDM is considered major compared to others like birth weight [41]. With regard to birth weight, it is remarkable that for the same birth weight (4000–4500 g), the prevalence of shoulder dystocia was 7% in nondiabetic women and 14% in diabetic women, and for a birth weight >4500 g, the risk was further increased (15% vs >50%) [42]. Moreover, the risk of brachial plexus injury is approximately 20 times higher when the birth weight is above 4500 g [43]. It is well known that macrosomia may complicate vaginal birth and the risk is increased when the fetus is atypically large like in GDM condition. Consequently, a prolonged labor in which the fetus might be stuck in the birth canal is frequent. Therefore, instrumental delivery (with forceps or vacuum) may be needed, and sometimes, an emergency cesarean section may be necessary. Furthermore, a greater risk of serious laceration and tear of the vaginal tissue may occur. In GDM pregnancies, macrosomia is often associated with polyhydramnios which may lead to an excessive uterus extension with high risk of uterine atony, resulting in postpartum hemorrhage. For the above-mentioned reasons in relation to excessive uterus extension and possible premature rupture of membranes, preterm birth (99%) composed of MI. However, the content of MI and DCI is significantly different in fat, muscle, and liver, according to the distinct functions of the isomers [7]. The MI concentration is higher in tissues with high glucose consumption, such as the brain and heart [6]. Indeed, MI-IPG is strictly linked to improving cellular glucose uptake by inducing GLUT4 translocation to the cell membrane [8], downregulating the adenylate cyclase enzyme and inhibiting the catabolism of free fatty acids from adipose tissues. In this case, MI is responsible for an increase in adipocyte insulin sensitivity [6]. By contrast, DCI-IPG concentration is higher in tissues committed to the storage of glycogen (i.e., liver, muscles, and fat) and lower in tissues with high glucose consumption. In particular, DCI-IPG is the main participant in glycogen synthesis, inducing glycogen synthase and pyruvate dehydrogenase (PDH) [8,9]. These differences are detected in assessing the MI/DCI ratio in tissues. Indeed, MI and DCI ratios have been investigated and found to play a significant role in several physiological processes [10]. The epimerase, which converts MI to DCI, has a tissue-specific activity [7] and is responsible for MI/DCI specific ratios in different tissues and organs [11]. In particular, this ratio is around 40:1 in the peripheral blood [12], about 20:1 in the thecal cells, and within the range of 70:1–100:1 in the follicular fluid of dominant follicles [4]. These ratios underline and suggest a specific role for inositols, even though not clearly defined yet.

DCI role in the hormonal signaling pathway Considering the hormonal action of inositols, different findings emerged throughout the years. Inositols, and in particular MI, participate in the follicle-stimulating hormone (FSH) signaling pathway. FSH binds to the FSH receptor (FSH) and MI is a second intracellular mediator which mediates the signaling pathway that regulates the proliferation and maturation of granulosa cells. In addition, MI and PLP-C mediate the release of InsP3, which increase the intracellular Ca2+ levels [13]. Fig. 2 gives help in visualizing the complex pathway. During the final steps of oocyte maturation, the binding of InsP3 to its receptor 1 (IP3-R1) that further releases calcium seems to have a key role in promoting the meiotic progression [14]. Moreover, MI-IPG is related to cytoskeleton [15] regulation and the FSH-induced production of anti-M€ ullerian hormone (AMH), which is responsible for FSH sensitivity in the follicle [16].

Fig. 1 Roles of myo-inositol (MI) and D-chiro-inositol (DCI) in cellular insulin-regulated glucose metabolic pathways. (From Laganà AS, Garzon S, Casarin J, Franchi M, Ghezzi F. Inositol in polycystic ovary syndrome: restoring fertility through a pathophysiology-based approach. Trends Endocrinol Metab. 2018;29(11):768–80. https://doi.org/10.1016/j.tem.2018.09.001.)

Fig. 2 Roles of inositol as second messenger in follicle-stimulating hormone (FSH)/luteinizing hormone (LH) signaling pathways within the ovary. (From Laganà AS, Garzon S, Casarin J, Franchi M, Ghezzi F. Inositol in polycystic ovary syndrome: restoring fertility through a pathophysiology-based approach. Trends Endocrinol Metab. 2018;29(11):768–80. https://doi.org/10.1016/j.tem.2018.09.001.)

Supplementation with D-chiro-inositol in women

In addition to this, MI induces the meiotic progression of oocytes into mature oocytes in mice models. In contrast, MI reduction within the ovaries hampers physiological oocyte maturation [17]. Conversely, FSH activates cAMP/PKA pathways, which upregulates aromatase and, subsequently, steroidogenesis. It has been demonstrated that DCI directly stimulates the production of testosterone in human theca cells [18]. In its DCI-IPG form, DCI participates in the expression of genes encoding steroidogenic enzymes in human granulosa cells, reducing the mRNA expression of both aromatase (CYP19A1) [18] and cytochrome P450 side-chain cleavage (P450scc) in a dose-dependent fashion [19]. Furthermore, DCI increases testosterone levels in the theca cells of women suffering from polycystic ovary syndrome (PCOS). This occurs because it acts as an insulin mimetic [18]. MI and DCI share some chemical properties but they appear to have quite different effects on metabolic pathways. MI, for example, seems to have a positive effect on aromatase expression, while DCI has been proven to inhibit it. Regarding the MI action on aromatase, there is a generally held hypothesis regarding the role of the second messenger InsP3, which is known to induce the aromatase activity in granulosa cells [20]. However, this opinion is actually a trend and not derived properly from the data. In particular, it could be useful to consider the MI and DCI action as a quantitative feedback on aromatase, where the MI/DCI ratio could be responsible for the final effect. In particular, a higher MI/DCI ratio should have a positive influence on aromatase activity in granulosa cells, increasing estrogen biosynthesis. Contrastingly, a lower MI/DCI ratio should reduce the estrogen levels and increase the androgen production in theca cells [21].

DCI supplementation DCI properties DCI has a known steroidogenic activity that can be distinguished in: 1. an indirect effect mediated by the insulin pathway; 2. an independent direct effect on steroidogenesis through downregulation of the aromatase gene expression and cytochrome P450 side-chain cleavage (P450scc) genes. Studies regarding DCI supplementation are mainly related to clinical validation of effects in women with PCOS. They often present insulin resistance and, after treatment, its reduction. In particular, DCI acts in the following ways. First, DCI is responsible for a decrease in androgens in the short term accompanied by a decrease in estrogen [22]. Secondly, DCI targets aromatase and testosterone synthesis. This causes androgen levels to increase once again [23]. DCI, when administered at higher doses, can therefore act in the short-term period by increasing androgen levels [24]. Taking previous studies into account [25], it seems logical to propose DCI supplementation in all conditions where a decrease in estradiol and an increase in testosterone are considered useful.



A clinical guide to inositols

PCOS Polycystic ovary syndrome (PCOS) is a clinical syndrome featuring chronic anovulation and hyperandrogenism, which can affect up to 10% of women of reproductive age. Etiopathogenesis is heterogeneous and not completely clarified [26–28]. PCOS classification has been thoroughly studied and health workers have found that the Rotterdam criteria are the most appropriate. The Rotterdam criteria were proposed in 2003 by the European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) [29] and consider at least two of three criteria in between: (1) clinical or biochemical hyperandrogenism; (2) menstrual irregularity; (3) polycystic ovaries characterized by ultrasound detection of 12 or more follicles 10 mL and in the absence of a dominant follicle. MI and DCI are currently being studied and used in the treatment of PCOS women, due to their insulin-sensitizing action. The idea of supplementing inositols in PCOS women is derived from two considerations: (1) the referred properties of inositols; (2) the need for reducing the amount of metformin. From these two points, a great deal of interest in the scientific literature has grown with an emphasis on studying the real efficacy of inositols in PCOS women. PCOS is generally characterized by insulin resistance, whose feature is responsible for a reduced MI-to-DCI conversion [30,31]. However, ovaries do not develop a reduced insulin sensitivity and, therefore, they are responsive to high insulin levels [32]. This condition impairs the correct and physiologic granulosa-to-theca layer ratio, causing an increase in the thickness of the theca when compared to the granulosa [33]. Hyperinsulinemia boosts the LH signaling pathway on theca cells, which leads to an increase in testosterone and, subsequently, estrogens. In addition to this, in the ovaries, the hyperinsulinemic status enhances the MI-to-DCI conversion in theca cells and decrease in the MI/DCI ratio, thanks to the overstimulation of epimerase activity [4]. In summarizing, in PCOS women, an altered MI/DCI ratio occurs with a decreased value. In contrast, in healthy women, MI is best represented. These data come from studies assessing the MI/DCI ratio in theca cells [4] and follicular fluid [34] of healthy women and women with PCOS. For this reason, it seems quite reasonable and logical to propose an appropriate and tailored treatment with MI and DCI for women with PCOS. Careful attention should be given to the timing of the treatment. In fact, the positive effect of DCI has been proven to be efficient in the short-term period. However, in the long-term period, DCI could reduce the aromatase action and potentially increase testosterone levels, thereby worsening the PCOS condition.

Supplementation with D-chiro-inositol in women

About 20 years ago, it was observed that in obese PCOS women, a 1200 mg/day DCI supplement significantly reduced the level of serum testosterone and increased ovulation. Moreover, it improved metabolism, reducing both blood pressure and triglycerides [5]. Subsequently, a new study increased the DCI dosage to 2400 mg/day. Somewhat surprisingly, the previous results were not confirmed, and the testosterone levels did not decrease [24]. However, regarding the stereoisomeric forms of inositols, studies have tried to find the correct MI/DCI ratio. The DCI supplementation alone is not adequate for PCOS women. Indeed, high dosages of DCI have been considered detrimental to ovaries and oocyte maturation [35]. Moreover, DCI is not converted into MI. This results in a reduction in the specific activity of MI. Subsequently, the loss of MI, and its intracellular form, correlates with insulin resistance. Facchinetti et al. [36] performed a metanalysis comparing the short-term effect of MI and metformin in relieving PCOS symptoms. In particular, in PCOS women no difference was found for MI and metformin in terms of fasting insulin, homeostasis model assessment (HOMA) index, testosterone, androstenedione, and sex hormone binding globulin (SHBG). By contrast, MI was associated with a lower risk of adverse events in comparison to metformin. For this reason, it seems quite reasonable to use MI in order to reduce metformin therapeutic dosage or improve its effect on improving insulin sensibilization [36]. Nordio et al. first described results comparing the administration of different MI/DCI ratios (0:1; 1:3.5; 2.5:1; 5:1; 20:1; 40:1, and 80:1) in PCOS women. The primary outcome investigated was ovulation, detecting the means of progesterone levels. The secondary outcomes were metabolic parameters such as FSH, LH, SHBG, E2, free testosterone, HOMA index, and basal and postprandial insulin. The best ratio tested was shown to be the 40:1 ratio, followed by 20:1 and 80:1. By contrast, the other combinations did not show any significant outcomes [37]. In particular, evidence suggests a combined 40:1 ratio with successful benefits in terms of phenotype and fertility improvement [38]. A recent metanalysis [39] considered 9 RCTs regarding the supplementation of MI alone or in association with DCI in the 40:1 ratio in PCOS women. The authors reported that inositol supplementation significantly reduced fasting insulin and HOMA index. Fig. 3 graphically shows the inositol effect on estrogen biosynthesis in granulosa cells. In PCOS women, an increase in epimerase activity due to insulin action determines a stereoisomeric shift from MI to DCI. The increase in DCI exerts its effect in downregulating aromatase and its activity. As a consequence, a lower amount of testosterone is converted into estradiol. In conclusion, the PCOS metabolic pathway results in an increase in androgen levels and a decrease in estrogen levels. Reasonably, a shift from MI to DCI needs supplementation with MI, in order to balance the inositol stereoisomeric equilibrium.


Fig. 3 Schematic representation of how inositols affect estrogen biosynthesis in granulosa cells of healthy and PCOS women. (From Unfer V, Dinicola S, Laganà AS, Bizzarri M. Altered ovarian inositol ratios may account for pathological steroidogenesis in PCOS. Int J Mol Sci. 2020;21 (19):7157. https://doi.org/10.3390/ijms21197157.)

Supplementation with D-chiro-inositol in women

Ovulation Anovulation is a condition that affects fertility and accounts for a significant proportion of all causes of infertility. In these patients, menstrual cycles occur but without ovulation. Moreover, continuous anovulatory cycles can have an effect on bone metabolism. In particular, it has been demonstrated that anovulatory cycles increase bone resorption. By contrast, ovulatory cycles reduce bone resorption during their luteal phase and the following cycle [40]. Anovulation has been classified by the ESHRE Capri Workshop Group [41] as follows: (1) Primary ovarian insufficiency: a condition where there is an absence or a severely reduced pool of primordial oocytes. It is characterized by amenorrhea and elevated FSH and LH serum levels. (2) Secondary ovarian insufficiency: a condition that is characterized by the lack of appropriate gonadotrophic stimulation of the ovaries. It can therefore be distinguished in: a. hypogonadotrophic hypogonadism (the WHO group 1). In this case, anovulation occurs since the levels of LH and FSH are below the threshold necessary in order to stimulate antral follicle development. Subsequently, estrogen levels are found to be low. b. normogonadotrophic anovulation (WHO group 2). In this scenario, anovulation occurs, but gonadotropin levels are normal. Different etiologies can counter this condition, but 91% of cases account exclusively for women with PCOS. In fact, PCOS is the most common endocrine disorder in women and is the principal cause of anovulation [42]. In anovulatory women from WHO group 2, the best-case scenario from an ideal treatment aims to induce ovulation. However, before triggering the ovulation itself with human chorionic gonadotropin (hCG), an appropriate therapy, which has the effect of restoring the hypothalamus-pituitary-ovary axis, is needed. Oral agents, which reduce the biosynthesis of estrogens and consequently decrease the negative hypothalamic feedback on gonadotropin-releasing hormone (GnRH) and follicle-stimulating hormone (FSH), are required [43]. Although selective estrogen receptor modulators, such as clomiphene citrate (CC), or third-generation AI, such as letrozole or anastrozole can be used, DCI has recently been found to be an important effector. Indeed, DCI has a dose-dependent effect in inducing ovulation. In particular, in PCOS women, a DCI dosage as high as 1200 mg/day for 6–8 weeks was found to be adequate in inducing ovulation in 44% of patients compared to the control group [44]. Moreover, this effect was independent of the BMI of patients [45]. Similarly, Bezzerra Espinola et al. recently presented a case report indicating that a 6-week treatment with 1200 mg/day DCI is able to induce



A clinical guide to inositols

ovulation in non-PCOS lean women. Ovulation was marked by a progesterone increase from 0.5 to 12 ng/mL [46,47]. The ovulation induction can be achieved by means of two different mechanisms: (1) the decreased activity of aromatase, which reduces the estrogen level. This result restores the normal hypothalamus-pituitary-ovary axis feedback, allowing the FSH secretion and subsequent antral follicle recruitment. (2) lower levels of insulin, which are seen to act in a two-fold way. First, the insulinstimulated biosynthesis of androgens is decreased. Secondly, the insulin-dependent activity of aromatase is hampered. For this reason, it seems quite understandable to use a combination of DCI with metformin. In particular, the final effect can be both a direct participation of DCI in reducing the estrogen levels and an indirect effect reducing the metformin dosage and subsequently possible side effects [48]. However, due to the androgen-increasing effect of DCI, longterm supplements with high dosages should be avoided. In particular, high dosages could have a potential negative impact on the ovary, as explained later in “The ovarian paradox” section. Indeed, data from Bevilacqua et al. suggest that high dosages of DCI induce histological PCOS-like phenotype in mouse models. This phenotype is characterized by cystic tertiary follicles with atretic oocytes or devoid of oocytes [47,49].

Endometriosis Endometriosis is a frequent gynecological pathology that affects both women’s quality of life and fertility and it is known to affect about 10% of women of reproductive age [50]. It occurs primarily during reproductive age and tends to fade out with menopause when estrogen production decreases. Endometriosis is characterized by the growth of endometrial tissue outside the uterine cavity. Generally, about 25% of affected women present no symptoms. However, in most cases, women suffer from dysmenorrhea, menorrhagia, pelvic pain, and chronic fatigue [50]. Moreover, as previously stated, endometriosis often impairs infertility which leads to a 10-fold lower possibility of conceiving when compared to a normal fertile couple. Regarding the histopathology of endometriosis tissue, previous findings enable us to find a possible target for DCI supplementation. In particular, the endometrial tissues responsible for endometriosis pathology have been proven to express high levels of aromatase [51]. This enzyme causes the booster of estrogen stimulation and the maintenance of a pro-inflammatory environment [52]. In particular, the production of estrogens from the endometrium makes the pathology sustain itself. The classic therapeutic approach lies in the reduction of estrogen levels, which hampers the endometrial growth and reduces the symptomatology. For this reason, therapeutic approaches with the aim of reducing the level of estrogens include letrozole and anastrozole, which significantly reduce pelvic pain, lesion size, and endometrioma volume [53]. Similarly, treatments with an insulin-sensitizing effect have been

Supplementation with D-chiro-inositol in women

studied and demonstrated to be efficient. Metformin, for example, reduces the inflammatory response, inhibits the aromatase activity, and allows the regression of endometrioma [54]. Herein lies the rationale for DCI supplementation. Since its role in inhibiting the aromatase, especially in high doses or long-term supplementation, it seems to have a logical and potential role in reducing the estrogenic environment and relief from the endometriosis symptomatology. Moreover, DCI supplementation may play a key role in reducing the side effects of the above-mentioned therapies by associating it with a multi-pharmacologic approach.

Uterine myomas Uterine myomas, also called fibroids or leiomyomas, are common benign tumors that grow from the smooth muscle cells of the uterus and affect between 20% and 30% of women of childbearing age. In most cases, myomas are asymptomatic and spontaneously vanish after menopause [55]. However, symptomatic myomas have been known to lead to pelvic pain, dysmenorrhea, and metrorrhagia, which impair women’s quality of life. Previous studies on myoma cells found that the enzyme aromatase is overexpressed compared with normal myometrium [56,57]. As a consequence, estrogen levels are increased, and progesterone receptors are overexpressed [58]. Nowadays, the gold standard treatment for symptomatic uterine myomas relies on a surgical approach [59,60]. However, an approach that focuses on the prevention of surgery-related complications is targeting the symptoms of myomas. In this context, ulipristal acetate (UPA), a selective progesterone receptor modulator (SPRM), was the first-line therapy for the treatment of myoma. The effect of this drug was to silence progesterone signaling and therefore reduce myoma growth [61]. However, since serious adverse effects were registered, which included liver failure, UPA was withdrawn from the pharmacologic market [62]. Parallel research studies focused on reducing estrogen levels [63]. In particular, a hypoestrogenic environment has been demonstrated to reduce the proliferation of uterine myoma cells [64] and decrease the size of uterine leiomyomas in postmenopausal women [65]. Medical treatment with letrozole, an aromatase inhibitor, has been found to be more efficient than hormonal therapy in reducing myoma volume. This has recently ignited growing interest in proposing the use of DCI for uterine myomas [66]. Firstly, DCI is a natural molecule, and adverse effects are expected to be lower than the previously administered drugs. Secondly, previously cited DCI properties can be effective and summarized in a two-fold way: (1) DCI induces aromatase downregulation which leads to decreased systemic estrogen levels; (2) DCI can negatively modulate aromatase expression directly in smooth muscle cells, lowering estrogen production in situ and slowing down the growth of tumors.



A clinical guide to inositols

For these known properties, DCI may reduce the estrogenic environment which in turn triggers the growth of uterine myomas. Secondly, it can alleviate signs and symptoms which negatively affect women’s quality of life. When taken together, these possible effects indicate that DCI can also be a potential adjuvant therapy prior to myoma surgery and can therefore reduce the possibility of surgery-related complications. However, well-designed RCTs are needed before making definitive conclusions. In particular, protocols addressing the dosage and frequency of DCI administration should be performed.

Endometrial hyperplasia Endometrial hyperplasia is defined as a precancerous lesion of the endometrium. This lesion shows a high cell proliferation rate that may result in endometrial carcinoma. Histopathologically, endometrial hyperplasia can be divided into typical and atypical forms. In the first case, only a modification in cell structure occurs. In the second case, more modifications are reported, including atypia and supernumerary nuclei [67]. Known risk factors for endometrial hyperplasia include age, nulliparity, high body mass index, anovulatory cycles, and high estrogen levels [68]. Among them, estrogens are critical since they trigger endometrial proliferation in 80% of cases. Of these, estradiol is the main cause of triggering proliferation [69]. Moreover, an increased expression of aromatase P450 has been found in endometrial hyperplasia cells in women with a background of polycystic ovary syndrome (PCOS) [70]. This expression leads to a local increase in estrogen levels with a subsequent growth stimulus. Aromatase inhibitors, like anastrozole, have been shown to play a key role in reducing local estrogen stimulus and revealing atrophic endometrium [71]. However, considering the potential side effects caused by letrozole administration and the studied effects of DCI, a potential role for this natural molecule can be found in treating endometrial hyperplasia. In fact, DCI downregulates aromatase and, subsequently, reduces estrogen levels. For this reason, DCI could be an efficient and effective supplement for reducing proliferation and, possibly, in the treatment of endometrial hyperplasia. What is clear is that well-designed RCTs are needed, also considering the unethical approach of administering only DCI in one group of patients. However, DCI supplementation with known and established therapies, such as letrozole, could be the first therapeutic approach in these patients.

Breast and endometrial tumors Breast and endometrial tumors have several risk factors in common. In particular, the majority of breast cancers and type-1 endometrial cancers overexpress the estrogen receptors. As a consequence, the risk of both types of tumors is higher in women with elevated systemic estrogen levels [72,73]. In addition, previous studies suggest that insulin and insulin-like growth factors (IGFs) may have a role in endometrial carcinogenesis, acting

Supplementation with D-chiro-inositol in women

in collaboration with estrogens [74]. Aromatase inhibitors are used in these hormonalsensitizing tumors due to their negative effect on estrogen production. In addition to this, they have been found to be effective in reducing the long-term recurrence of estrogendependent cancer and the related mortality rate [75]. However, as cited earlier, chronic treatment with aromatase inhibitors can lead to possible side effects. In particular, the consequent hypoestrogenic status can affect women’s quality of life and bone resorption. For this reason, DCI supplementation can have a key role in implementing aromatase inhibitor activity by downregulating aromatase. Probably, as stated earlier, for endometrial hyperplasia, DCI could act primarily as a supplement to established hormonal therapies.

Mood disorder Anxiety and depressive disorders are the most common of all psychiatric disorders [76]. Epidemiological analysis shows their widespread prevalence and a gender difference correlation [77]. In particular, the number of females who are afflicted by mood disorders is more than double of that in males [78]. This gender gap indicates a potential role for gonadal hormones in the etiology of anxiety and depressive disorders. In fact, previous studies have demonstrated that women are more likely to experience mood disorders, anxiety, and depression during hormonal fluctuations, such as puberty, menopause, perimenstrual, and postpartum periods [79]. Regarding the possible hormonal role in etiology, the relationship between testosterone levels and mood disorders has been elucidated in males with hypogonadism [80]. Similarly, testosterone plays a key role in women’s behavior. In particular, its concentrations have been found to be more than 10 times higher than that of estrogens in some areas of the brain, such as the hypothalamus [81]. For this reason, testosterone treatment has been considered eligible for restoring normal libido in women with low testosterone levels associated with a mood disorder and reduced libido [82]. Intriguingly, previous studies have already demonstrated the beneficial effect of testosterone supplementation. In particular, in a study conducted more than 30 years ago, healthy menopausal women were treated with testosterone combined with estrogens or estrogens alone. The study showed that women in the first group felt better in terms of mood, feeling more composed, elated, and energetic when compared to the group that received only estrogen. Moreover, the latter showed higher levels of circulating estradiol [83]. Additionally, in perimenopausal women with climacteric mood disorders, testosterone administration positively influenced their quality of life [84,85]. Taking these data together and considering the known properties of DCI, it is possible to propose its supplementation in women affected by mood disorders. In fact, aromatase downregulation can raise testosterone levels and restore women’s quality of life and sexual well-being. Future studies are needed since no DCI supplementation protocols have so far been performed. However, the DCI safety profile and properties can be used as a propellent for DCI usage as a testosterone adjuvant or, possibly, a substitute.



A clinical guide to inositols

Ovarian hyperstimulation syndrome (OHSS) Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that occurs during controlled ovarian stimulation (COS) in assisted reproductive treatments (ART). This syndrome is characterized by an increase in vascular permeability and extravascular fluid storage which can have a strong detrimental effect on women’s health. OHSS can have a wide spectrum of clinical symptoms from mild (prevalence of 20%– 33%), such as abdominal discomfort, to severe with life-threatening complications (prevalence of 3%–8%). Globally, the incidence of OHSS is in a decline. A stable decrease has been reported after its peak incidence in the 1990s, with an incidence of severe OHSS of 0.2%–1%. In those years, during COS, the main goal was to obtain a higher number of oocytes [86,87]. Actually, a retrospective cohort study described a decline in OHSS incidence from 3.6% in 2005 to 1% in 2009 [88]. The syndrome originates from an excessive ovarian response to gonadotropins used for follicle stimulation, resulting in multifollicular growth. In addition to this, the human chorionic gonadotropin (hCG) triggers the OHSS onset [89]. Timing can divide the syndrome into early and late onset, which is mainly sustained by endogenous hCG. The vascular endothelial growth factor (VEGF) is addressed as the main effector of the syndrome since it increases the permeability of ovarian blood vessels, which brings a fluid shift from the intravascular space to mainly the peritoneum [90,91]. The first medical approach should be prevention, and this requires an assessment of risk factors and strict monitoring of clinical markers. Of these, unusually elevated estrogen levels are generally considered red flags [92]. Moreover, when OHSS is suspected, cancellation of the ovarian stimulation cycle should be considered, even though this could have an economic and psychological burden on women [93]. Contrastingly, strategies aiming at reducing the incidence of OHSS include postponing (coasting) of the hCG ovulation trigger injection until the estradiol levels decrease to under a safe threshold [94]. Regarding a possible therapeutic approach, estrogen reduction can be achieved with a short-term. letrozole treatment before hCG administration. This strategy has been shown to significantly reduce the incidence of OHSS in high-risk women undergoing COS [95,96]. Intriguingly, long-term treatments of more than 30 days with metformin during COS have been proven to significantly reduce the risk of OHSS [97]. Taking all these results into consideration, a potential role of DCI arises in the prevention of OHSS. In particular, the DCI efficacy can be consequent to: (1) its indirect effect in mediating the insulin signaling pathway. (2) the downregulation of aromatase and, subsequently, the estrogen levels. This natural compound could likely be used as an adjuvant treatment, especially in highrisk patients. For this reason, its role should be elucidated with further studies with the aim of finding a proper indication for preventing OHSS onset.

Supplementation with D-chiro-inositol in women

The ovarian paradox DCI’s role in ovarian physiology is still under debate [98]. As stated above, the MI:DCI ratio in the follicular fluid has been found to be 100:1 [4]. This finding can ex ante suggest an inositol role in the ovaries which is mainly played by MI. However, in order to find possible implications of the two stereoisomeric forms, studies arising from the assisted reproductive treatment (ART) field come to our aid. In ART, evidence has been produced regarding the oocyte influence of both MI and DCI. Indeed, the analyses of MI: DCI ratio in the follicular fluid of couples undergoing IVF revealed that lower ratios, corresponding to higher DCI contents, define poor-quality blastocysts. By contrast, MI with higher contents features better-quality blastocysts. Moreover, MI administration in patients undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) showed promising results, with increased ovarian response in terms of oocytes retrieved and improved oocyte quality (with less vesicle germ and degenerated oocytes) [99–102]. From these data, a new term has recently been coined, the “ovarian paradox.” This term explicates the schism existing from the positive role of DCI for gynecologic purpose and its negative effect on ovaries. More clearly, in various conditions or pathologies, DCI has a beneficial role as stated earlier, such as in endometriosis, uterine myoma, and hormonal cancer management, due to its aromatase inhibition role. However, ovarian activity is impaired by DCI, especially in high doses. Since the two inositol stereoisomers show different actions and present opposite results, actual evidence does not support the DCI supplementation alone in order to increase ovarian response in ART. Moreover, these data can be a key clue in directing the DCI supplementation indications. When an aromatase inhibition is requested, DCI could exert its beneficial role. In other cases, for example in PCOS, appropriate attention should be given to the MI:DCI ratio in order to limit the DCI negative effect on the ovaries. In particular, combined MI and DCI oral supplementation at the ratio of 40:1 is proposed as an appropriate treatment to restore normal ovarian function in PCOS women and improve metabolic state at the same time.

Safety Currently, studies on the DCI safety profile have not been performed yet. However, data from studies can suggest a safety profile for DCI supplementation. In particular, DCI administered 1.2 g/day does not present any relevant adverse side effects. However, as stated earlier, the DCI supplementation impairs the steroid metabolism and can induce an increase in androgen levels. This condition can worsen the PCOS symptomatology and should be taken into consideration by health workers. Moreover, attention should be given when considering the “ovarian paradox” in DCI administration.



A clinical guide to inositols

Conclusion DCI has been extensively studied in its physiopathology and it is known to downregulate the gene expression of aromatase. This property can have a positive therapeutic effect that can be distinguished in both increasing androgen and reducing estrogen levels. For this reason, a positive effect exists for DCI on different conditions affecting women’s health or quality of life. In particular, a key solution to induce ovulation can be found in DCI since its role in helping the FSH axis restoration. In addition, due to its activity in aromatase downregulation and subsequently the estrogen level reduction, DCI supplementation could be useful in every condition where aromatase inhibitors are indicated. In particular, endometriosis, endometrial hyperplasia, endometrial and breast tumors, uterine myoma, and mood disorders can all benefit from DCI activity. This possible beneficial effect could therefore help in reducing the amount of drug that needs to be administered, especially in chronic therapeutic protocols. Well-designed randomized controlled studies are needed in order to assess these beneficial targets. In addition, the natural properties of DCI suggest that a safe profile and a low incidence of side effects can be expected. However, consideration should be given to the “ovarian paradox,” a term that reflects a negative action of DCI in the ovaries during assisted reproductive treatments. Specifically, high dosages of DCI supplementation can impair ovarian activity, resulting in lower oocytes retrieved and with lower quality after controlled ovarian stimulation. This should be carefully considered when starting a DCI supplementation.

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A clinical guide to inositols

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Application of myo-inositol and D-chiro-inositol in andrological issues Annarita Stringaroa, Maurizio Nordiob, and Monica Vazquez-Levinc a

National Center for Drug Research and Evaluation, Italian National Institute of Health, Rome, Italy Department of Experimental Medicine, University “Sapienza”, Rome, Italy c Institute of Biology and Experimental Medicine (BYME, CONICET-FIBYME), National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina b

Introduction Inositols are nowadays considered an important family of natural cyclohexane that can strongly impact male fertility. The most abundant isomer in nature, myo-inositol (MI), constitutes an important element of membrane phospholipids, together with its less represented relative, D-chiro-inositol (DCI). The latter is synthesized in the human body from MI through an insulin-dependent epimerase enzyme [1,2]. They contribute to different physiological functions, including osmoregulation, protein phosphorylation, chromatin remodeling, and gene expression. Their phosphorylated derivatives, instead, act as second messengers in several pathways, and in sperm cells, they are involved in transduction mechanisms responsible for cytoplasmic calcium level regulation [3]. Indeed, they are mostly known as second messengers of insulin, even if they play different roles in this process. Overall, the correct cellular ratio between MI and DCI is fundamental to optimize glucose uptake and its metabolism [1,2]. Moreover, they are involved in the signaling of gonadotropins, as in clinical trials they demonstrated a gonadotropin-sensitizing effect. MI and DCI, in fact, reduce the amount of circulating follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively [1,2]. Likewise, they have different impacts on steroidogenesis, as MI seems to support estrogen production, while DCI inhibits androgen conversion to estrogens, thus promoting androgen accumulation. Accordingly, their clinical applications in men should cover different therapeutic areas.

Role of myo-inositol in male fertility In nature, inositol is expressed into different chemical compounds: free form, bound to phospholipids and in the form of phytic acid. The second form is contained in animal tissues while the third one is in plants. So, inositol can be both biosynthesized and taken A Clinical Guide to Inositols https://doi.org/10.1016/B978-0-323-91673-8.00012-1

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A clinical guide to inositols

with diet through whole grains, wheat germ, citrus fruits, and meats [4]. The intestinal absorption of free inositol is achieved, thanks to active transport, temperature- and pH-dependent of SMIT family. It is an active transport with a negative Na+ concentration gradient dependent on both concentration and energy, which allows uptake and accumulation [5]. Different inositol phosphates are also metabolized into MI by inositol-phosphatephosphatase 1 (MINPP1) and then transported into cytosol. Cytosolic glucose can also be used to synthesize MI through myo-inositol-phosphate synthase (MIPS) [6]. Also, an integral membrane protein of the SMIT family carries one molecule of MI and two of Na+ in the cells. This carrier is encoded by the SLC5A3 gene, controlled by osmoregulatory elements [7,8]. The expression of the SLC5A3 cellular transporter was found in several tissues, including testis and epididymis. Moreover, mRNA expression was also detected in Sertoli cells. In addition, Sertoli cells subjected to hypertonic conditions showed a significant increase in SLC5A3 and an increased MI uptake. The SLC5A3 expression in Sertoli cells and MI increased expression and uptake in hypertonic conditions suggest MI as a key regulator of osmolarity in the male gonad [6,9]. At the cellular level, inositol is present under four types of molecular combinations: 1. embedded in phospholipids and anchored to the membrane. In this chance, it participates in the phosphoinositide signal cascade and transduction of FSH signal in Sertoli cells, an additional MI mechanism of action which, by sensitizing Sertoli cells to FSH, might potentially improve sperm parameters, in control of levels of intracellular calcium, in maintaining the potential of a membrane [10]. Specifically, close to the endoplasmic membrane, it undergoes a series of phosphorylations, which lead to the formation of various metabolites; among which, of relevant importance, phosphatidylinositol 4,5 bisphosphate (PIP2) [11] and then phospholipase C (PLC) metabolizes PIP2 into 1,2-diacylglycerol (DAG) and inositol (1,4,5)-triphosphate (IP3) that activates IP3 receptor (IP3R) and induces calcium release from the endoplasmic reticulum reservoir; 2. component of glycosyl phosphoinositides [6]; 3. as polyphosphates [6]; 4. as simple inositol [12]. MI possesses a pivotal role as an intracellular second messenger through the regulation of intracellular Ca2+ levels and regulates different mechanisms such as sperm motility, capacitation, and acrosome reaction. Activation of intracellular transmission systems, which necessarily lead to an increase in cytoplasmic Ca2+, determines the consequent increase also at the mitochondrial level. At this point, Ca2+ stimulates the oxidative metabolism, inducing the production of ATP in response to the requested cellular energy [13]. To work at best, this system needs a good functional state of the mitochondria and consequently of high MMP. Recently, it has been shown how the high MMP correlates closely not only to an improvement in the fertilizing capacity of spermatozoa but also to

Myo-inositol and D-chiro-inositol in andrological issues

an improvement in sperm motility [14]. Interestingly, some recent in vitro studies highlighted an important role of myo-inositol, connected with increased MMP and sperm motility. All these findings give the important notion that myo-inositol, through the regulation of intracellular levels of Ca2+, is capable of influencing in the better way the fertilizing capacities [15]. Moreover, it is well known that MI, acting as a second messenger, regulates the FSH activity implicated in the control of Sertoli cell number and function [16] and is essential to sustain normal spermatogenesis [17]. Different results highlighted that high concentrations of FSH and LH in serum were found in the case of low sperm concentration [18,19]. On the other hand, MI increased the levels of inhibin B, a glycoprotein secreted from the testis as a product of Sertoli cells involved in the regulation of FSH secretion by a negative feedback mechanism [20,21]. In men, either with normal or altered spermatogenesis, a strong inverse correlation has been reported between inhibin B and FSH levels [22,23]. In the course of years, different in vitro studies examined whether MI may improve sperm parameters. The samples of OAT patients treated with inositol 2 mg/mL showed the absence of the amorphous material implicated in excessive viscosity of the seminal fluid and reduced sperm motility. Furthermore, the mitochondria were morphologically more similar to control samples, with less damage involving mitochondrial cristae [24]. Similar results were obtained by incubating the samples with 2 mg/mL of MI for 2 h, and MI increased significantly the number of spermatozoa with high MMP and decreased significantly the number of those with low MMP in OAT patients with respect to a placebo [25]. Another similar in vitro study showed an increase in the percentage of spermatozoa with progressive motility in both normospermic men and patients with OAT, also demonstrating an improvement in motility in the first group, associated with a significant increase in the percentage of spermatozoa with high MPP. Moreover, after incubation with MI, the total number of spermatozoa recovered after swim-up improved significantly in both groups [15]. Since studies showed that sperm motility is directly associated with fertilization rate, even in IVF procedures, there were different studies that evaluate the impact of MI in IVF procedures [26–28]. The presence of MI resulted in both an increase in proliferation activity and developmental rate of in vitro cultured early mouse embryos, representing a substantial improvement in culture conditions, measured by daily progression through cleavage stages, blastocyst production and expansion, and blastomere number at 96 h post-fertilization [29]. In a prospective, bicentric, randomized study, 78 ICSI cycles were divided into two groups, and spermatozoa were treated with myo-inositol or with placebo. The fertilization rate and percentage of grade A embryos on day 3 were significantly higher when spermatozoa were treated in vitro with myoinositol versus placebo [30]. Then, another study utilized myo-inositol in vitro to verify its effect on semen quality in both normal and OAT patients undergoing in vitro



A clinical guide to inositols

fertilization (IVF) with respect to standard sperm medium. In vitro incubation of seminal liquid carried out using myo-inositol at a concentration of 15 mg/mL showed improved progressive motility in both normospermia and OAT subjects [31]. Similar results were collected by patients’ ejaculates with hyperviscosity or OAT patients. Incubation with MI improved sperm progressive motility in high viscosity samples compared to the control group and in OAT patients [32]. Since the freezing process is quite stressful for all types of cells, another in vitro study evaluated the efficacy of MI on a number of parameters in order to improve the capacitation protocols commonly used in assisted reproductive technology (ART). Treatment of samples with MI showed an increase in the sperm total and progressive motility in both fresh and thawed samples, so MI can be efficiently and safely used in the laboratory practice and for the preparation of semen samples in ART. Specifically, MI registered positive results on progressive motility, normal morphology, lipid peroxidation, and DNA fragmentation [33,34]. Also, MI in vitro supplementation to ejaculated human sperm from infertile men resulted in a significant increase in the cryo-survival rate (CSR) in samples with abnormal pre-freeze sperm parameters [35]. Concerning the improvement of motility, a recent study evaluated the linear and nonlinear progressive motility. Such study found a significant increase in linear progressive and a significant reduction in nonlinear progressive motility after incubation with MI [36]. Improving semen quality, in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. In this regard, myo-inositol, based also on its antioxidant properties, is reported to be effective in improving sperm quality and motility in patients undergoing assisted reproduction techniques. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, MMP, and sperm motility. The same results are reported in in vivo studies. Sperm and metabolic parameters related to male infertility are evaluated following a dietary supplement based on MI. In a study conducted on idiopathic infertility, patients demonstrated that MI rather than placebo can be useful to improve sperm parameters, such as the percentage of spermatozoa with acrosome reaction, spermatozoa concentration, total count, and progressive motility. Furthermore, an improvement was obtained in hormonal parameters, thanks to a reduction in FSH and LH and a concomitant increase in inhibin B concentrations [37]. In another study, samples from healthy and oligoasthenospermic (OA) patients were analyzed before and after the administration of MI, revealing a significant increase in sperm concentration in the OA patient group and a significant increase in sperm count in the healthy patient group [38]. Moreover, in asthenospermic patients with metabolic syndrome, a mix of MI and antioxidant molecules showed significant improvements in sperm parameters (concentration, motility, and morphology), hormonal profile (testosterone, estradiol, LH, and SHBG), and metabolic (HOMA index) parameters [39]. In addition, 85.32% of asthenospermic patients achieved a significant improvement in sperm motility [40]. For the first time, researchers investigated the effect of MI on

Myo-inositol and D-chiro-inositol in andrological issues

cholesterol efflux, a hallmark of capacitation. They underlined an increase in cholesterol efflux in the spermatozoa of patients with OAT treated either in vitro or in vivo with a blend of nutraceuticals, containing mainly MI. The same study also found an increase in the activity of G6PDH, associated with an increase in glucose metabolism through pentose phosphate pathway (PPP), both in normal patients and patients with OAT [41].

Role of D-chiro-inositol in male fertility Steroidal hormones are pivotal in male reproductive tissues. Testosterone (T) and estradiol (E2) are the two principal hormones involved in male physiology. Both of these hormones contribute to the development of primary and secondary sexual characteristics during adolescence, participating also in the homeostatic processes during adulthood [42]. The biosynthesis of T occurs via different steps in the biological process called steroidogenesis. Eventually, the conversion of T to E2 involves enzymatic processes that keep an adequate amount of both, maintaining the balance. Hence, both functional and transcriptional regulations of the participating enzymes are of primary importance. Specifically, the conversion of T in E2 is mediated by an enzyme called aromatase, also known as estrogen synthetase (gene CYP19A1). Such enzyme is widely expressed in multiple tissues, including testes, granulosa cells, placenta, bones, breasts, and adipose tissues [43]. The latter is particularly rich in aromatase content [44] and produces clinically relevant amounts of circulating estrogens [45]. As a consequence of the pivotal role that such enzyme plays, alterations in its activity lead to a hormonal imbalance. In fact, excessive aromatase activity results in reduced levels of T and increased concentration of E2 [43]. DCI participates in the regulation of hormone production, strongly influencing steroidogenesis. Indeed, growing evidence exists concerning the role of DCI in the different steps of steroidogenesis. In this regard, the first data highlighted that DCI modulates insulin-induced androgen biosynthesis in the ovaries [46]. Subsequent evidence underlined that DCI increases T levels either directly, stimulating T biosynthesis in human ovarian theca cells [47], and indirectly, decreasing the CYP19A1 aromatase gene expression in granulosa cells [48]. The DCI therapeutical profiles are similar to those of the aromatase inhibitors, a class of drugs that allosterically block the active site of the enzyme [1,2]. However, the mechanisms of action are quite different from those pharmaceuticals. In fact, DCI lacks the capability to obstruct the active site of the enzyme, participating in fine regulation of the process through the transcriptional inhibition of the aromatase. A recent study [42] specifically designed to demonstrate the clinical relevance of such molecule provided the first insights into the clinical activities of DCI in this sense. In this study, 10 healthy volunteers took 600 mg of DCI twice per day for 1 month, reporting no adverse events. All these men had normal hormonal profiles at baseline, which were kept in the physiological range even after the treatment. Nonetheless, significant changes in these values



A clinical guide to inositols

occurred, thanks to DCI. Indeed, 9 out of 10 volunteers experienced an increase in T, reporting also decreased E2 following the treatment. Accordingly, E1 levels decreased while DCI improved DHEAS. Moreover, DCI restored normal values of glycemia in almost all hyperglycemic subjects. This study firstly highlighted that the downregulation of aromatase induced by DCI represents a valid therapeutical mechanism in males when the purpose is to increase T. Hypogonadal males constitute a particular subpopulation that needs an increase in T. These men experience an age-dependent loss of T deriving from an impairment in the hypothalamic-pituitary-testicular (HPT) axis. Treatment nowadays preferred in the case of hypogonadism is testosterone replacement therapy. However, concerns exist about the potential adverse events of such therapy in older men, focusing on the prostate and cardiovascular system. In light of this, Nordio and colleagues carried out a pilot study to test whether DCI could represent suitable treatment, inhibiting aromatase expression to induce T accumulation [49]. In such pilot study, the authors enrolled 10 men aged 65–75 with decreased sexual desire, weakened morning erections, erectile dysfunction, and low levels of serum testosterone without relevant diseases other than hypogonadism. Their findings highlight that DCI significantly improved both metabolic and hormonal profiles. Specifically, DCI induced an increase in androgens, namely T and androstenedione, while decreasing E2 and estrone. Therefore, DCI treatment increased the T/E2 ratio, inducing a shift toward physiological levels.

Role of oxidative stress and antioxidant treatments The antioxidant system in the male fertility Oxidative stress can exert detrimental effects on spermatozoa into the epididymis where these cells complete their maturation during transit. This attack leads to negative changes that can lead to infertility [50,51]. In this phase, spermatozoa are physiologically exposed to reactive oxygen species (ROS) generated by metabolic pathways, glycolysis, or oxidative phosphorylation (OXPHOS) to activate intracellular mechanisms involved in physiological functions such as sperm capacitation-associated events, such as acrosome reaction [51]. Furthermore, mature spermatozoa are haploid with a highly compacted nucleus that cannot transcribe and does not have the capacity to respond to stress stimuli [52,53]. So, a spermatic-protective antioxidant system in the semen constituted by both enzymatic and nonenzymatic factors with antioxidant capacity is fundamental to protect the sperm against ROS. This antioxidant enzyme system in the semen is called an enzyme triad comprising superoxide dismutase, catalase, and glutathione peroxidase. In addition, the high proportion of polyunsaturated fatty acids (PUFAs) in the sperm plasma membrane makes spermatozoa especially susceptible to suffering lipid peroxidation (LPO) that can generate a negative alteration of membrane fluidity, mitochondria dysfunction, abnormal morphology, reduction of sperm viability, premature acrosome exocytosis,

Myo-inositol and D-chiro-inositol in andrological issues

and defective signaling events during capacitation leading to fertilization failures [50,53]. If on the one side ROS are harmful to spermatozoa, on the other side they can be useful for some functions necessary for the fertilization processes but when the quantity of ROS exceeds the antioxidant defense mechanisms, the result is an oxidative stress [51,52]. When accumulated in excess, activated oxygen species attack all organic components (lipids, carbohydrates, and proteins, including nucleic acids) that can lead to cell death and the effects can be LPO or DNA damage and consequently a reduction in sperm motility, morphology, and viability which are associated with lower sperm fertility [53,54]. Of all cellular compartments, mitochondria are the major source of ROS due to the reactions that occur during the mitochondrial breathing phase for the production of ATP [55]. These mitochondria generate ATP through the respiratory electron chain and oxidative phosphorylation, which are based on transferring electrons from inner mitochondrial membrane complexes to oxygen and pumping of protons to the intermembrane space. In this context, especially at the level of complexes I and III, they can release superoxide and hydroxyl radicals into the matrix and intermembrane space [54]. Lipid peroxidation of the sperm plasma membrane is one of the targets of choice of ROS frequently associated with low sperm quality and infertility [56]. During LPO, are formed a lot of reactive molecules such as propanol, hexanol, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) highly reactive and that may attack other nearby PUFAs, initiating a chain reaction with harmful effects that eventually disrupts membrane fluidity [50]. These molecules have the potential of causing DNA damage and modifying proteins. For example, the aldehyde compounds inhibit some antioxidant enzymes which reduce the activity of glutathione peroxidase [54]. Furthermore, 4-HNE is capable of inducing mutations of the mitochondrial DNA and forming adducts with mitochondrial proteins that lead to mitochondrial dysfunction [57]. Other products of LPO can bind mitochondrial proteins of the electron transport chain and consequently decrease mitochondrial membrane potential, decrease ATP production, and decrease sperm motility [58]. Indeed, in the last years, researchers highlighted that mitochondrial membrane potential can be used as a measurement of sperm quality and above all a strong correlation with sperm motility [59]. Furthermore, in different andrological pathologies, the loss of mitochondrial membrane potential is also associated with several mechanisms of cell death, mainly the activation of caspases, preceded by permeability transition pore (PTP) formation or by coordination of Bcl-2 proteins [59]. Excessive generation of ROS in the reproductive tract is not only affecting the fluidity of the sperm plasma membrane but also the integrity of DNA in the sperm nucleus. To concern DNA damage, it is reported principally in base modifications, DNA strand breaks, and chromatin cross-linking [52]. Fragmentation of sperm DNA (SFD), whether single-stranded or double-stranded, is the most recognized form of DNA



A clinical guide to inositols

damage to the sperm nucleus. Sperm DNA oxidative stress studies over the past decade have shown that nucleosides, particularly guanosine and adenosine, are very sensitive to oxidation. High sperm nucleus fragmentation has been clearly associated with an increased risk of miscarriage, poor embryonic quality, and implantation failure [53,58]. As previously mentioned, sperm genetic material such as nuclear chromatin is highly stable and compact. The normal DNA structure is capable of decondensing at an appropriate time transferring the packaged genetic information to the egg without defects in the fertilization process [52]. Oxidative stress can also promote nuclear decondensation, increasing the DNA susceptibility to being damaged by free radicals, which will thus have easier access to the entire sperm genome [54]. DNA repair is not possible during nuclear condensation in the epididymis and the last opportunity to repair DNA damage is by the human oocyte, which is a critical step in embryo development, ever depending on maternal age. The ds-DNA break results in genomic instability and apoptosis in the absence of repair. Apoptosis via multiple cell death signaling and regulatory pathways is known as a physiologically programmed cell death due to DNA fragmentation. ROS-induced ds-DNA breaks can result in apoptosis [58].

Antioxidant supplementation for male fertility Different studies highlighted the importance of supplementation with antioxidant substances in both in vitro and in vivo studies. It studied the free radical scavenging proprieties of folic acid because its constituents, pyrazine and pterin, can easily be reduced by the hydrated electron to the corresponding hydroderivatives in the pyrazine ring of the molecule [60,61]. Scientific studies reported that in folate-deficient cells the concentration of lipid peroxidation is increased probably owing to its capacity to activate NF-κB, a controller of apoptosis process [62]. Different studies and a recent systematic review analyzed the supplementation of folic acid in subfertile males, and the results are very similar. In summing, the results showed an improvement in the number of spermatozoa after 3 months of supplementation with 15 mg of folic acid and improved endocrine parameters by stimulating the Sertoli cells, the main producers of inhibin B. The serum concentration of inhibin B relates to sperm concentration because it reflects the correct activity of Sertoli cells, thus representing a marker of good spermatogenesis in humans [63–67]. L-carnitine has a crucial role to transport acetyl and acyl groups into the mitochondrial membrane and for this reason is essential for the metabolism of long-chain fatty acids [68,69]. It is detectable as free or acetylated forms in epididymal tissue, seminal plasma, and spermatozoa [66,70]. Moreover, L-carnitine shows the activity of free radical scavengers, especially to superoxide anion [71]. Indeed, in a preclinical study on an animal

Myo-inositol and D-chiro-inositol in andrological issues

model, L-carnitine demonstrated preservation of the acrosome integrity of sperm and inhibition of apoptosis [72,73] or positive results in sperm vitality, motility, count, and reduction in ROS [74–76]. Furthermore, different randomized placebo-controlled studies on subfertile patients showed a significant improvement in semen quality, specifically in sperm concentration and motility [77–82] and reduction in the percentage of sperm with incorrect morphologies [83]. L-arginine actively participates in the formation of sperm and prevents the peroxidation of membrane lipids involving nitric oxide (NO), a short-lived free radical, synthesized by a class of NADPH-dependent enzymes called nitric oxide synthases (NOS) [84–86]. These enzymes catalyze the conversion of L-arginine to L-citrulline and NO [82]. In vitro studies on sperm function have controversial results. Some evidence indicate that low concentrations of NO increase human sperm capacitation [87]. Others suggest opposite results [88,89]. Moreover, NO inactivates superoxide anions and can reduce lipid peroxidation by inactivating superoxide. Based on the ability of L-arginine to increase the generation of NO, it is clear that L-arginine protects spermatozoa against lipid peroxidation, thanks to its capacity to biosynthesize nitric oxide [90]. Unfortunately, a few quantities of studies were published on L-arginine and sperm parameters. In vitro study demonstrated an improvement in sperm motility and a decrease in lipid peroxidation according to its mechanism of action [86,91]. A further study investigated the clinical efficacy in infertile men giving the same result on sperm motility and moreover concentration and morphology [92–94]. N-acetyl-cysteine (NAC) is widely known as a mucolytic agent due to its ability to break the disulfide bonds in the high-molecular-weight glycoproteins of mucus, reducing the viscosity. Moreover, several in vitro studies reported efficient antioxidant activity principally related to three different mechanisms [95]: (1) a direct antioxidant effect on certain oxidant species including NO2 and hypohalous acids (HOX) [96]; (2) an indirect antioxidant effect as a result of the ability of NAC to act as a precursor of cysteine, important for the glutathione synthesis [97]; (3) a breaking effect on disulfides and the ability to restore thiol pools, which in turn regulate the redox state [98,99]. Further studies involving animal models to evaluate NAC efficacy demonstrated that it has a protective effect against DNA damage improving sperm parameters and seminal vesicle weight to minimize leukemia treatment [100,101]. In vivo studies demonstrated that NAC can optimize sperm parameters such as count, motility, and abnormal morphology. Moreover, the hormonal profile showed an improvement in lowering FSH and LH levels and increasing testosterone levels. DNA fragmentation showed significant decreases, total antioxidant capacity (TAC) significantly increased and malondialdehyde (MDA) decreased, showing an inverse correlation



A clinical guide to inositols

between TAC and MDA [102–104]. The same results were confirmed in a group of varicocele patients because the production of ROS is one of the main events associated [105]. In another in vivo study after NAC treatment, the serum total antioxidant capacity was greater and the total peroxide and oxidative stress index were lower in respect to the control group. These beneficial effects resulted from reduced reactive oxygen species in the serum and reduced viscosity of the semen [106]. As previously anticipated, a clinical trial involving men with metabolic syndrome highlighted the efficacy of the supplementation with MI and antioxidant compounds to improve semen quality. Indeed, the authors prescribed treatment with MI, selenium, and L-arginine. They report a decrease in the HOMA index, SHBG, and estradiol, with a concomitant increase in LH, free and total testosterone [39].

Conclusions Inositols are natural compounds that exist in all the body districts, with each stereoisomer playing specific and diverse roles. If, on the one hand, MI is pivotal for the FSH signal, on the other hand, there is a lack of evidence on the physiological roles of DCI. Nonetheless, DCI proved useful to increase testosterone values both in healthy and hypogonadal males. On the contrary, MI supplementation, both alone and in combination with other antioxidants, proved useful to improve the fertility of infertile men. Despite the promising data, there is still a need for further high-quality studies to assess the roles of the two stereoisomers and their therapeutical usefulness.

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Myo-inositol and D-chiro-inositol in andrological issues

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A clinical guide to inositols

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Myo-inositol for subclinical hypothyroidism and potential prevention of thyroid tumors Daniele Barbaroa, Giuseppina Porcarob, and Salvatore Benvengac,d a

U.O. Endocrinology ASL Nord Ovest, Tuscany, Italy Women’s Health Centre, Terni, Italy c Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy d Interdepartmental Program of Molecular & Clinical Endocrinology and Women’s Endocrine Health, University Hospital, Messina, Italy b

Introduction Thyroid-stimulating hormone (TSH) is the major physiologic regulator of growth and functioning of the epithelial (endodermic) component of the thyroid: the follicular cells, also termed thyrocytes. Further to the complex regulation of thyroid hormone synthesis (starting from the stimulation of iodide uptake) and secretion into the bloodstream, TSH also governs replication and differentiation of the thyrocytes. The maturation of pituitary-thyroid axis and subsequent stimulation of thyroid growth and functioning by TSH start from the third trimester of gestation, since early thyroid development and thyroglobulin (Tg) synthesis at the onset of folliculogenesis is TSH-independent. Accordingly, impaired TSH synthesis or TSH signaling due, for instance, to inactivating mutations of the TSH receptor (TSHR) may cause thyroid hypoplasia but not athyreosis. Once secreted by the anterior pituitary, TSH binds to its cognate receptor (TSHR), which is located on basolateral side of the plasma membrane of thyrocytes. As known, TSHR is a G-coupled membrane receptor that activates second messengers. These effects are largely mediated by the increase of intracellular cyclic AMP (cAMP) concentrations that follows the TSH-TSHR binding. In addition to cAMP (and cAMP-dependent protein kinase A [PKA]), TSH activates the phospholipase C (PLC)-dependent inositol phosphate Ca2+/diacylglycerol (DAG) pathway, with formation mainly of inositol 1,4,5-triphosphate (IP3). IP3 increases the concentration of intracellular Ca2+ by favoring its release from the endoplasmic reticulum. This duality of messengers also applies to the TSHR-stimulating activity of the circulating immunoglobulins associated with Graves’ disease [1–3]. In addition to IP3, as explained with greater details elsewhere in this book, other inositols (ISs) are well-known second messengers whose role is gaining increasing interest in

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A clinical guide to inositols

the regulation of several metabolic and endocrinological pathways. ISs exhibit a hexahydroxy cyclohexane structure, with 9 different stereoisomers having different degrees of phosphorylation, myo-inositol (MI), and D-chiro inositol (DCI), which are the most studied stereoisomers, and overall they can be phosphorylated at seven possible positions. IS can be synthesized de novo from glucose-6-phospate or introduced by diet and rarely incorporated in the cellular membranes in the phosphorylated forms named phosphoinositides (PPIs) which represent about 1% of the total phospholipids. The different sites of phosphorylation of the polyol cyclohexane bring about different roles in cellular functions [4]. Single phosphorylations are especially involved in the regulation of endocytosis and cell trafficking. On the other hand, specific lipid kinases can modify the extent of phosphorylation, with IS polyphosphates appearing to act mainly as second messengers for cell replication. As already stated, one of the most studied ISs is IP3, which acts as one of the second messengers for some peptide hormones, including TSH. Vice versa, other polyphosphate ISs (3.4 and 3.4.5) mainly mediate the action of growth factors on thyrocytes. After binding of growth factors to their receptors, inositol 3.4 is released from cell membrane; after additional phosphorylation(s), inositol 3.4 can bind to domain inactivating AKT, a serine/threonine-specific protein kinase also known as protein kinase B. Activating mutations in proteins of the PI3K/Akt/mTOR pathways are implicated in tumorigenesis, including thyroid cancer (Fig. 1). For these reasons, the abovementioned IS polyphosphates (3.4 e 3.4.5 polyphosphates) could exert a protective (antitumoral) role. Higher phosphorylated IS can be also generated, but their role remains to be completely clarified [1–4]. Since ISs influence functioning and replication of the thyrocytes, below we will discuss the effect of supplementation with MI, which is the most abundant IS, in hypothyroidism, with emphasis on subclinical hypothyroidism (SCH) and on thyroid tumorigenesis.

Myo-inositol and hypothyroidism A decreased/absent physiological functioning of thyroid caused by different intrinsic factors is associated with a condition termed primary hypothyroidism, while conditions leading to hypofunction stemming from lesions at the hypothalamus and/or pituitary level are commonly recognized as central hypothyroidism. The degree of the thyroid gland impairment can be minimal/moderate (called SCH or initial hypothyroidism) and is compatible with circulating free thyroxine (fT4) levels still within the reference limits but with increased serum TSH levels (most frequently up to 10 mU/L). However, the thyroid gland impairment can be severe, resulting in low concentrations of serum fT4 and high concentrations of serum TSH, a condition termed frank or overt hypothyroidism. Hypothyroidism can translate into different clinical implications depending on the age of onset and severity, the rate of development, and some special conditions, either

Myo-inositol for subclinical hypothyroidism and thyroid tumors basolateral membrane











IP3 other IPs


I cell growth proliferation survival


TG transcription

T3 T4



apical membrane



Iodine organification

Fig. 1 Schematic representation of synthesis and secretion of thyroid hormones in the thyrocyte. Myoinositol (MI) is involved in the phosphorylative cascade which is important in the organification mechanism, leading to thyroid hormones’ production.

physiological (pregnancy) or pathological (specific comorbidities). Table 1 summarizes the causes of primary hypothyroidism. Thyroid dysfunction is underestimated in the population, with hypothyroidism prevailing over hyperthyroidism, as demonstrated by a recent meta-analysis concerning the European population. The mean prevalence of undiagnosed hypothyroidism and undiagnosed hyperthyroidism was 4.94% (95% CI, 4.75%–5.13%) and 1.72% (1.66%–1.88%), respectively,



A clinical guide to inositols

Table 1 Causes of noncongenital (acquired) primary hypothyroidism in nonendemic regions. Autoimmune

Chronic autoimmune thyroiditis (AIT), also known as chronic lymphocytic thyroiditis or Hashimoto’s thyroiditis Iatrogenic

After thyroidectomy After radioactive iodine therapy After external radiotherapy for neck and head cancer Drug-related (amiodarone, lithium, cytokines, cancer therapy with tyrosine kinase inhibitors [TKIs], cancer immunotherapy with monoclonal antibodies, antithyroid drugs, and other drugs)

with a female preponderance in either dysfunction. Importantly, 80.1% of the undiagnosed thyroid dysfunction was subclinical. The prevalence of undiagnosed total, overt, and subclinical HT in females was 6.40%, 0.80%, and 5.86%, respectively, whereas for males, it was 3.37%, 0.30%, and 3.45%, respectively [5]. As recently reviewed, “At present, most of the international societal guidelines advise that treatment decisions should be individualized based on patient age, degree of serum TSH elevation, symptoms, cardiovascular disease risk, and other comorbidities.” Accordingly, there could be room for the use of nutraceuticals [6,7]. Most data on nutraceutical are about selenium properties. Selenium led the way in such nutraceutical setting, with the organic and more bioavailable form L-selenomethionine (SeM) highlighting better results than the inorganic form (sodium selenite). However, the organic forms can theoretically cause accumulation of Se, so that the usually advised dosage should not be overcome. Members of the Association of Medicals Endocrinologist (AME) were invited to participate in a web-based survey investigating the use of selenium in different clinical conditions. Among respondents, 85.2% considered the administration of selenium for thyroid disease (58.1% rarely/occasionally and 27.1% often/always), and 79.4% prescribed the molecule for chronic autoimmune thyroiditis (AIT) (39.1% sometimes and 40.3% often/always). Approximately two-thirds of the respondents considered using selenium in cases of SCH, and approximately 40% suggested using selenium for patients with AIT who were planning pregnancy or were already pregnant [8,9]. This national survey prompted a survey among members of the European Thyroid Association (ETA). A minority of responding ETA members stated that the available evidence warrants the use of Se in HT, but a majority recommended it to some extent, especially to patients not yet receiving L-thyroxine (LT4) [7–9]. On these premises, the use of nutraceutical virtually with no side effects and with the purpose to restore a more physiological condition is now gaining interest just in the early phases of thyroid failure [10–16].

Myo-inositol for subclinical hypothyroidism and thyroid tumors

Based on its important role in cell regulation [17], MI can theoretically find an application in SCH regardless of SCH etiology; in fact, there is literature on the MI which can have a generic thyroid-protective effect of MI as shown by data in vitro and on animals in vivo. Searching on PubMed using the string “Inositol and Hypothyroidism,” we found 32 papers, most of which are of clinical nature and concerning SCH in AIT. We analyze the pathophysiological rationale for the use of MI in AIT and in general in SCH, and we report data on clinical use of MI supplementation. Starting this discussion from the possible role of MI in AIT-related SCH, we should keep in mind the role of MI in TSH action [1–3,18]. As already mentioned, after the binding to its receptor, TSH activates two pathways. One pathway involves cAMP as second messenger which has a trophic effect regulating cell proliferation, differentiation, and thyroid hormone secretion. The second pathway, associated with phospholipases C, involves IS as second messengers; it is mainly implicated in H2O2 generation and H2O2-mediated iodination of certain tyrosine residues of Tg, although an effect on PI3K/Akt/mTOR pathways is now recognized. cAMP cascade is stimulated at low concentrations of TSH, while IS cascade is stimulated at high concentrations of TSH [5]. Hence, ISs are essential for thyroid hormone production finally cooperating with cAMP cascade at the level of the synthesis [1,3]. However, the role of IS can be at a double level, since some in vitro studies suggest that inositol IP3 and other IS polyphosphates like 3.4,5 triphosphate are implicated in the immune response [19]. For these reasons, the dietary supplementation of MI can have an emerging interest, with some original papers showing that MI can restore normal values of TSH in AIT-associated SCH [20–22]. Of course, three specific points need to be addressed. The first is whether MI supplementation can have a role in mechanistically antagonizing the onset and progression of SCH. The second concerns the optima dose and duration of such supplementation. The third point concerns MI supplementation in certain settings, such as gestation. To date, four main original studies, summarized in Table 2, are available. The first paper [23] was a prospective randomized double-blind study. Forty-eight women with mean age of 38 years were enrolled. All subjects had AIT, and the TSH levels were increased in the range between 4.01 and 9.99 mU/L. Patients were treated with MI plus SeM or with SeM alone. The primary end point was the normalization of TSH levels; the secondary end points were the decrease of serum TPOAb and/or TgAb. Patients were randomized into two groups: one group (n ¼ 24) received only SeM 83 micrograms for 6 months while the other group (n ¼ 24) received SeM 83 micrograms and MI 600 mg for the same period. TSH statistically decreased in the second group as ultrasound echogenicity improved, while AbTPO and AbTg improved in both groups. A subsequent paper [24] on a greater number of patients presenting TSH levels in a narrower range confirmed the superiority of the MI + SeM association. In the last work, 168 patients were enrolled in a prospective randomized trial and subdivided into two groups (1:1 ratio) receiving as in the previous work SeM or SeM plus MI. TSH was in the high



A clinical guide to inositols

Table 2 Clinical studies on the effectiveness of combination MI plus SeM in patients WTH AIT. Author (ref.)

Patients and design

Main findings

Nordio and Pajalich [25]

Prospective, randomized, doubleblind 6-month-duration study. Of 48 patients (all women) with AIT, 24 received daily 600 mg MI plus 83 μg SeM, while 24 received daily 83 μg SeM

Nordio and Basciani [24]

Prospective, randomized, 6-month-duration study. Of 168 patients with AIT, 84 patients (75 women, 9 men) received daily 600 mg MI plus 83 μg SeM while 84 (74 women, 10 men) received daily 83 μg SeM Prospective not randomized. 21 patients with AIT treated with MI 600 mg plus SeM 83 twice a day for 6 months

Significant decrease of TSH (31%), TPOAb (44%), and TgAb (48%) in the MI plus SeM group. Unchanged TSH and significantly decreased TPOAb (42%) and TgAb (38%) in SeM alone group. Significantly greater rate of improvement of ultrasound echogenicity in the MI plus SeM group compared to the SeM alone group (24/24 vs. 10/24) Decrease of TSH (4.22 0.6 vs 3.26 0.89 mU/L) and increase FT4 (0.93 +/ 0.15 vs 1.06  014) in the MI plus SeM group after 6 months

Ferrari [26]

Pace [27]

Observational, retrospective, 12-month-duration study. 101 patients (86 women 15 males) with AIT divided into three groups: untreated (controls, n ¼ 29); treated with SeM alone (83 μg/day; n ¼ 29); and treated with SeM (83 μg/day) plus MI (600 mg/day; n ¼ 43)

After six months, TSH declined (1.35  0.70 Vs 2.01 0.86 mU/mL) and TgAb declined significantly. Chemokine CXCL10 and TPOAb decreased although not at significant level TSH increased significantly (+35%) in controls but decreased significantly in SeM-treated (31%) and SeM + MItreated (38%) groups, in the latter group already at 6 months (26%). The rate of SCH (which at baseline was greater in the two treated groups (51.7% and 81.4%) compared to controls (34.5%)) increased in controls and SeM-treated patients (75.9% both), but remained stable in the SeM + MI-treated patients (+79.1%) Thyroid hypoechogenicity decreased similarly

AIT, autoimmune thyroiditis/Hashimoto’s thyroiditis; MI, myo-inositol; SeM, selenomethionine; AbTg, thyroglobulin autoantibodies; AbTPO, thyroperoxidase autoantibodies.

Myo-inositol for subclinical hypothyroidism and thyroid tumors

normal range or slightly elevated (3–6 mU/L). A significant reduction of serum TSH (plus increase of fT4) levels was demonstrated in the second group over 6 months. These interesting papers lack a control group of patients without any supplementation, so that it is not fully clear if the effect of MI is directly depending on MI administration itself or derives from a synergistic activity with SeM. The third paper [26] aimed to study the possible role of MI plus SeM in the progression of HT in euthyroid patients. Twenty-one patients were enrolled and treated with SeM plus MI (83 μg/600 mg) twice a day for 6 months. TSH decreased in all patients being the decrease more remarkable in patients with high/normal TSH values. Classical serum markers of thyroid autoimmunity (TPOAb and TgAb) decreased. Moreover, in this paper, also the chemokine CXCL10 (also known as interferon γ inducible protein) was assessed. CXCL10 declined, although not significantly. These results suggest an effect of MI plus SeM on the progression of hypothyroidism both in terms of TSH and of a decrease in the burden of autoimmunity. Also, in this paper, control groups of SeM alone and no supplementation are lacking. The data of the third work are corroborated by an in vitro study of the same authors which contain not only a control group, but also a group treated with MI alone [28]. This study showed a favorable effect of MI, SeM, or their combination on the hydrogen peroxide-induced oxidative stress of peripheral blood mononuclear cells from patients with AIT. As known, AIT is characterized by an intra-thyroid lymphocytic infiltration and overproduction of cytokines, including chemokines, by both lymphocytes and thyrocytes. Chemokines have been associated with aggressiveness of AIT and, therefore, with deterioration of thyroid functionality. AIT is also characterized by enhanced oxidative stress. Furthermore, in the experiments, it was assumed that peripheral blood mononuclear cells (PBMCs) mirror thyroid-infiltrating lymphocytes. PBMCs from HT or control women were exposed to hydrogen peroxide (H2O2)-induced oxidative stress with subsequent addition of MI (0.25, 0.5, 1.0 μM), SeM (0.25, 0.5, 1.0 μM), or their combination (0.25 + 0.25, 0.5 + 0.5, 1.0 + 1.0 μM). Both in controls and in HT women, the combination of MI plus SeM resulted in the greatest decrease in both PBMC proliferation and secretion into the medium of two chemokines (CXCL10/IP10. CCL2 and CXCL9/MIG). Such effects were dose-dependent. The fourth study [27] was a retrospective trial where Pace and colleagues analyzed 101 patients (86 women and 15 men) with AIT, divided into three groups: 29 control (no treatment), 29 treated with SeM 83 μg, and 43 treated with SeM plus MI 83 micrograms/600 mg once a day for 12 months, with TSH measurements complemented by measurements of fT4 and free triiodothyronine (fT3). Moreover, ultrasonography was performed to evaluate the hypoechoic pattern. TSH values increased at 6 and 12 months in control group while decreased in SeM group at 12 months and in MI plus SeM at 6 months. The percentage of SCH increased in control group and in SeM group (although not significantly) while the percentage of SCH was unchanged in SeM plus



A clinical guide to inositols

MI group. This retrospective study confirms the data from the three previous prospective studies, suggesting a synergistic role of SeM plus MI, although it would have been desirable to have a group treated only with MI. Overall, all these data suggest a potential role of MI in restoring euthyroidism in AIT with SCH. Additionally, the AIT-associated deterioration of thyroid function appears to be improved by MI in AIT, and this can be a favorable effect on both the thyroid cells as thyrocytes and lymphocytes. It is not completely clear if the effect of MI is synergistic with SeM, so that further studies are encouraged. However, MI appears to be a novel way to approach AIT with or without SCH, while it is more difficult to speculate the potential role of MI in SCH due to causes of SCH other than AIT. As already stated, in vitro data from human thyroid cells and animals highlight a protective effect of MI, and this suggests a potential role in the other form of SCH. Some additional pathogenetic considerations could suggest a possible role of MI alone or associated with iodine in areas with suboptimal iodine intake [29]. Clinical studies should be conducted in this setting. Because of the association of AIT with thyroid cancer, in an ad hoc section of this chapter, we will summarize the effect of MI alone, SeM alone, and MI plus SeM on CXCL10 and CCL2. Table 2 summarizes the clinical studies.

Myo-inositol and subclinical hypothyroidism in pregnancy Pregnancy has a deep impact on the thyroid gland. During gestation, the thyroid gland increases in size by 10% in iodine replete countries, but by 20%–40% in areas of iodine deficiency. Production of the thyroid hormones, T4 and T3, increases by nearly 50%, in conjunction with a 50% increase in the daily iodine requirement [30]. These physiological changes support the physiological growth and development of fetus. During the first trimester, women exhibit high concentrations of serum human chorionic gonadotropin (hCG) which are accompanied by reduced circulating TSH levels. HCG has a thyrotrophic activity, and it can bind to the TSHR, due to their structural similarity, thus stimulating the synthesis and secretion of thyroid hormones. Because of the negative feedback of thyroid hormones on the hypothalamus and pituitary, TSH levels fall. Furthermore, during gestation, there is an augmented serum concentration of the thyroxine-binding globulin (TBG), an estrogen-dependent protein that acts as the major plasma carrier of thyroid hormones, with an associated increased concentration of total T3 and T4 [31]. Another crucial factor that can alter TSH levels in pregnancy is iodine availability. From early pregnancy, renal clearance of iodine is reported to increase, in parallel with the increase in glomerular filtration rate. This would explain some epidemiological data supporting a positive correlation between TSH level and urinary iodine concentration. This may gravely impact on the thyroid responsiveness to TSH [32,33].

Myo-inositol for subclinical hypothyroidism and thyroid tumors

Thyroid hormones are essential for the fetus, which during the first trimester depend completely on their transplacental passage. Thus, maternal thyroid hormones are particularly important for the neurodevelopment of the fetus. While normalization of TSH occurs by the second trimester, fT3 and fT4 levels remain slightly lower until the second and third trimesters. Furthermore, up to 18% of all pregnant women are TPOAb- or TgAb-positive. Data suggest that TPOAb positivity adversely modulates the impact of both the maternal and fetal thyroid statuses. The positivity to thyroid antibody independently increases the risk of thyroid dysfunction during the postpartum period [8,25,33]. Insufficient evidence exists to conclusively determine whether LT4 therapy decreases the risk for pregnancy loss in TPOAb-positive euthyroid women who are newly pregnant. However, administration of LT4 to TPOAb-positive euthyroid pregnant women with a prior history of fetus loss may be considered, given its 7 potential benefits in comparison with its minimal risk. In such cases, 25–50 μg of LT4 is a typical starting dose for the treatment [25]. For all these reasons, accurate assessment of maternal (and fetal) thyroid function during pregnancy remains difficult, and interpretation of laboratory testing differs from the nonpregnant patient. Serum TSH is considered the most accurate biochemical index of thyroid status. Regarding the diagnosis and management of thyroid disease in pregnancy, the 2011 American Thyroid Association (ATA) Guidelines recommended 2.5 μIU/mL as the upper limit of normal TSH in the first trimester, 3.0 μIU/mL in the second trimester, and 3.5 μIU/mL in the third trimester. However, the revised 2017 ATA Guidelines suggest using normative reference ranges obtained on the local population [34]. SCH defined as an increase of TSH level above these normal values, although maintaining normal fT4, is a common pregnancy-related thyroid disorder affecting the 3%–5% of pregnant women worldwide. Guidelines released by several scientific societies, including the Endocrine Society (ES), the American Thyroid Association (ATA) jointly with the American Association of Clinical Endocrinologists (AACE) and most recently by the European Thyroid Association (ETA), agree on the recommendation that SCH, as well as overt hypothyroidism, should be treated with LT4 replacement, in order to reduce pregnancy-associated risks to the fetus. Moreover, some studies, but not all, suggest that SCH could compromise the cognitive function of the offspring. The [34] Guidelines concluded that the evidence indicated an increasing risk of pregnancy-specific complications, most notably pregnancy loss and preterm delivery, in relation to elevated maternal TSH concentrations [34]. Early fetal loss occurs naturally in about 30% of pregnancies, and several studies have suggested a relationship between miscarriage and higher maternal TSH levels. In particular, there is a higher miscarriage rate in women with TSH concentration between 2.5 and 5.0 mU/L than in those with concentrations below 2.5 mU/L (6.1% vs 3.6%), as well as an increased risk of complications in women with SCH and AbTPO positivity.



A clinical guide to inositols

Furthermore, in thyroid antibody-positive women who are euthyroid, TSH levels increase progressively as pregnancy advances, namely from a mean of 1.7 μIU/mL in the first trimester to 3.5 μIU/mL in the third trimester, with approximately 19% of women presenting a TSH value above the upper limit at delivery. It seems reasonable to recommend or consider LT4 treatment for specific subgroups of pregnant women with SCH, and the strength of such recommendation should differ depending on TPOAb status. This recommendation also requires that any pregnant women with an elevated TSH concentration must also be evaluated for her TPOAb status. In making the recommendation, the task force acknowledges the very low risk associated to initiating low-dose LT4 treatment. A dose of only 50 μg/d is typically required for effective treatment of SCH women [34]. Also timing of intervention may play an important role in the effectiveness of intervention. Data demonstrate an overall reduction in pregnancy complication rate in women treated in the first trimester, but not in those treated in the second trimester, and that starting treatment immediately after the first prenatal visit decreases the premature birth rate. Particular attention should also be paid to euthyroid women with positive thyroid antibodies, as in these women, the TSH values increase progressively with the progress of gestation, with about 19% of women having a TSH value above the upper limit at delivery. Such women could become the target of nondrug therapies aimed at maintaining euthyroidism during pregnancy [35,36]. As stated above, numerous publications indeed demonstrated the beneficial effects of MI plus SeM treatment against SCH and AIT, both in vitro and in vivo. Thus, MI supplementation in pregnancy could be taken into consideration to prevent or treat SCH [16,37]. MI, as widely stated above, is involved in cell signaling, and, as second messenger, it regulates TSH and other hormones as follicle-stimulating hormone (FSH) and insulin activities [17,36,38]. It seems that the increase of cellular MI availability ameliorates TSH sensitivity of the thyroid follicular cells. Therefore, this might explain the effect of MI in maintaining the TSH levels at normal values also throughout patient’s pregnancy. In 2018, Porcaro and Angelozzi demonstrated the efficacy and the safety of MI plus SeM supplementation in pregnancy euthyroid women [39,40]. The study enrolled women between the ages of 18 and 40, with a body mass index between 19 and 25 kg/m2, and a single intrauterine pregnancy and TSH levels between 1.6 and 2.5 μIU/mL were enrolled. Women with abnormal thyroid function, history of miscarriage, or preterm delivery, and women with morbid obesity or undergoing any antithyroid treatment were excluded. A prospective study on pregnant women, from the first trimester until delivery, was carried out. Checks of TSH, fT3, and fT4 were performed at the first, second, and third trimesters (T0, T1, and T2). Women were divided into two

Myo-inositol for subclinical hypothyroidism and thyroid tumors

groups: group A (n ¼ 17) were treated with 600 mg MI plus 83 μg Se until delivery, and group B (n ¼ 16) received no treatment. Treatment started at the 10th week of gestation until the end of pregnancy. Primary outcome was the maintenance of normal TSH levels throughout pregnancy. Secondary outcomes included normal fT3 (2.57–4.43 pg/mL) and fT4 (0.93–1.70 ng/dL) levels throughout pregnancy. The results demonstrated a prevention against SCH thanks to the treatment (group A), with a stabilization of TSH, fT3, and fT4. TSH levels remained virtually unchanged in the treated group throughout pregnancy. One dropout was recorded in this group because TSH levels raised up to 3.00 μIU/ml in the second trimester, and she initiated treatment with LT4. All the other 16 patients continued the nutraceutical treatment until delivery. In the control group (group B), 3 dropouts were observed at the second trimester, and 2 further patients had high levels of TSH in the third trimester, started treatments with L-T4. This study highlighted that normal TH values were maintained in 94.1% of pregnant women in the treated group, compared to 68.7% in the control group, with a statistically significant difference. These findings are perfectly in line with previous clinical evidence that highlighted the beneficial effect of the supplementation of MI plus SeM in restoring the euthyroid state in patients diagnosed with SCH [39,40]. Obviously, the safety of MI in pregnancy was also confirmed by this study and by several studies conducted on the use of MI in gestational diabetes and in the prevention of spina bifida, despite the fact that MI was already widely used in other diseases related to pregnancy [13,41]. The supplementation of MI plus SeM appears to prevent thyroid hormone fluctuations while maintaining the euthyroid state in almost all pregnant women, so that MI plus SeM could help pregnant women to avoid maternal-fetal complications related to the possible onset of SCH [41]. The role of MI plus SeM in pregnant patients with AIT or SCH should be confirmed, even though such confirmatory trials would be hindered by ethical concerns, because in pregnant women with SCH, treatment with LT4 is mandatory [1,3,13]. However, MI could be of special interest in all cases in which TSH appears to be normal in the early pregnancy, but there can be some minor concerns regarding a possible development of SCH. Specifically, the up-to-date picture of clinical results reported in literature reveals that the supplementation MI plus SeM is useful to manage SCH conditions. Encouraging data from studies on this nutraceutical and thyroid disease sustain the use of MI to prevent the development of SCH in pregnancy.

Myo-inositol in thyroid tumorigenesis Thyroid cancer is the most common endocrine cancer, and its incidence is increasing worldwide even in the pediatric population. It is not the purpose of this work to discuss all the risk factors for thyroid cancer, but two points should be considered [42–47].



A clinical guide to inositols

The first is that AIT may predispose to the differentiated thyroid cancer, particularly to its leading histotype (papillary thyroid cancer [PTC]). In contrast, solid and wellknown is the association between AIT and the rare thyroid lymphoma [45,48–51]. The second point is the possible role in favoring thyroid cancer conferred by increased serum levels of TSH. A meta-analysis [52], involved 28 studies, a total of 42,032 subjects and 5786 thyroid cancer cases. For TSH levels 1.0 mU/L, the odds ratio (OR) for thyroid cancer was 1.16 per milliunits per liter. Studies controlling for autoimmunity reported the lowest OR (TSH below 2.5 mU/L, OR 1.23 per milliunits per liter; TSH 2.5 mU/L, OR 0.98 per milliunits per liter). The meta-analysis concluded that higher serum TSH concentration is associated with an increased risk of thyroid cancer and that thyroid autoimmunity may partially explain the association [53–55]. Although intriguing, in our opinion, these last data present a poor theoretical rationale also, because in AIT, the predisposition to papillary cancer appears more related to the same AIT condition with respect to the high TSH levels. However, the protective role of MI on the progression of AIT could represent a benefit [56,57]. More limited, and mostly based on case reports, is the evidence for AIT being a risk factor for medullary thyroid cancer. Table 3 reports a short classification of thyroid cancer and tumors, in line with the WHO classification. Thyroid cancer pathogenesis fits the general complex mechanism of neoplasia, which implicates a stimulus to proliferation and a block of apoptosis and senescence, in the background of multiple genetic mutations and epigenetic factors which can be both the cause and the effect [58]. Cell proliferation is governed by complex biochemical pathways, including those triggered by interaction of hormones with membrane receptors [59]. In some case, a mutation of oncogenes can produce a constitutive activation of some key enzymes; in Table 3 Thyroid tumors.

Carcinoma of follicular epithelium: - Papillary thyroid cancer - Follicular thyroid cancer - Poor differentiated thyroid cancer (insular or poor differentiated papillary cancer) - H€ urthle cell carcinoma - Anaplastic thyroid cancer Carcinoma of parafollicular origin: medullary thyroid cancer Other neoplasia: - Follicular adenomas - Thyroid tumors at uncertain malignant potential - Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) - H€ urthle cell adenoma

Myo-inositol for subclinical hypothyroidism and thyroid tumors

Table 4 Most frequent mutations and rearrangements in thyroid tumors derived from follcular cells.

Follicular cancer

Poor differentiated and anaplastic cancer

Follicular adenoma

+(K601E) ++


++(V600E) ++








 + + +

++  ++ ++




Main molecular alterations

Papillary cancer: classic and tall cells

Follicular variant of papillary cancer



–, very rare (30%).

other case, we can observe an inactivating mutation of an oncosuppressor gene. The mutations can involve membrane receptor or intracellular pathway leading to abnormal proliferation rate. In the introduction, we have already reported the possible role of IS in the complex regulation of intracellular proliferative pathways. Table 4 reports the main oncogenes and tumor-suppressor genes whose mutations are implicated in thyroid tumorigenesis. However, the roles of these mutations are incompletely understood. The most studied mutation is the BRAF-V600E one, whose importance as a crucial single mutation in cancer aggressiveness is still debated. Vice versa, a poor prognosis could derive from the contemporary presence of BRAF-V600E and TERT promoter mutations. Apart from RAS genes mutation and BRAF V600 E mutations, which are mutually exclusive, some oncogenes of the MAPK and PI3K/AKT cascades can cooperate in tumorigenesis. Thus, there could be space for the use of MI. Indeed, some studies have shown a potential benefit of MI in the oncology setting [17,60]. Overall, the role of MI can be at multiple levels. MI and especially its hexophosphate (MIP6) form can have a broad anticancer activity. Notably, these compounds show effects at different levels: (1) induction of G1 phase arrest and inhibition of phase S electively in cancer cells; (2) inhibition of PI3K/AKT pathway; (3) indirect anticancer effect by modulation of insulin activity; and (4) antioxidant effect. Particularly, the effect on PI3K/AKT pathway could work also to antagonize the activation of the cascade involving other oncogenes.



A clinical guide to inositols

To date, most in vitro and in vivo data are focused on colon cancer and breast cancer in which MI has been also useful in improving side effects and quality of life of patients receiving chemotherapy. Although the above-mentioned theoretical presuppositions appear promising, clinical studies supporting the use of MI in thyroid cancer are lacking. However, an interesting study [29] in thyroid nodules presenting low/intermediate risk of malignancy at ultrasonography is worthy of mention. This retrospective study enrolled patients with SCH or borderline high TSH levels and with thyroid nodules classified as class I and/ or II by AACE, ACE, AME guidelines. In these guidelines, class I means low-risk nodules, and class II means intermediate-risk nodules. The treatment group was of 18 patients, and they received MI plus SeM (600 mg/83 μg) once a day for 6 months while the control group of 16 patients received no treatment. The two groups were comparable for serum TSH levels, and nodule size, age, and BMI. The primary end point was the size of the nodule, and the secondary end points were TSH values, number of nodules, elasticity score, presence of calcifications, and vascularity. After 6 months, the size of nodules, the number of total nodules, and the elasticity score decreased significantly, as did serum TSH values. A limitation of this work is that we cannot infer if the decrease of the size and of the number of nodules is due to a direct effect of MI plus SeM on growth or it is an indirect effect, just mediated by the decline of serum TSH. However, because the decrease of TSH was mild (