How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

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Referencias bibliográficas:

1. Valko M, Leibfritz D, Moncol J, Cronin M, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:44 - 84.
2. Sugino N. Reactive oxygen species in ovarian physiology. Reprod Med Biol. 2005;4:31 - 44.
3. Espey L. Ovulation as an inflammatory reaction--a hypothesis. Biol Reprod. 1980;22(1):73 - 106.
4. Nakamura Y, Tamura H, Takayama H, Kato H. Increased endogenous level of melatonin in preovulatory human follicles does not directly influence progesterone production. Fertil Steril. 2003;80:1012 - 6.
5. Paszkowski T, Traub A, Robinson S, McMaster D. Selenium dependent glutathione peroxidase activity in human follicular fluid. Clin Chim Acta. 1995;236:173 - 80.
6. Acuña-Castroviejo D, Escames G, Venegas C, Díaz-Casado M, Lima-Cabello E, López L, et al. Extrapineal melatonin: sources, regulation, and potential functions. Cell Mol Life Sci. 2014;71:2997 - 3025.
7. Tamura H, Takasaki A, Miwa I, Taniguchi K, Maekawa R, Asada H, et al. Oxidative stress impairs oocyte quality and melatonin protects oocytes from free radical damage and improves fertilization rate. J Pineal Res. 2008;44:280 - 7.
8. Carroll J, Jones K, Whittingham D. Ca2+ release and the development of Ca2+ release mechanisms during oocyte maturation: a prelude to fertilization. Rev Reprod. 1996;1:137 - 43.
9. Chiu T, Rogers M, Law E, Briton-Jones C, Cheung L, Haines C. Follicular fluid and serum concentrations of myo-inositol in patients undergoing IVF: relationship with oocyte quality. Hum Reprod. 2002;17:1591 - 6.
10. Pesty A, Lefèvre B, Kubiak J, Géraud G, Tesarik J, Maro B. Mouse oocyte maturation is affected by lithium via the polyphosphoinositide metabolism and the microtubule network. Mol Reprod Dev. 1994;38:187 - 99.
11. Pacchiarotti A, Carlomagno G, Antonini G, Pacchiarotti A. Effect of myo-inositol and melatonin versus myo-inositol, in a randomized controlled trial, for improving in vitro fertilization of patients with polycystic ovarian syndrome. Gynecol Endocrinol. 2016;32:69 - 73.
12. Unfer V, Raffone E, Rizzo P, Buffo S. Effect of a supplementation with myo-inositol plus melatonin on oocyte quality in women who failed to conceive in previous in vitro fertilization cycles for poor oocyte quality: a prospective, longitudinal, cohort study. Gynecol Endocrinol. 2011;27:857 - 61.

RESÚMENES DE ARTÍCULOS RECOGIDOS EN LA BIBLIOGRAFÍA
1.Valko M y cols.
Int J Biochem Cell Biol. 2007;39:44 - 84.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS, e.g. nitric oxide, NO(*)) are well recognised for playing a dual role as both deleterious and beneficial species. ROS and RNS are normally generated by tightly regulated enzymes, such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. Overproduction of ROS (arising either from mitochondrial electron-transport chain or excessive stimulation of NAD(P)H) results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA. In contrast, beneficial effects of ROS/RNS (e.g. superoxide radical and nitric oxide) occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically, various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-establish or maintain "redox balance" termed also "redox homeostasis". The "two-faced" character of ROS is clearly substantiated. For example, a growing body of evidence shows that ROS within cells act as secondary messengers in intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. This review will describe the: (i) chemistry and biochemistry of ROS/RNS and sources of free radical generation; (ii) damage to DNA, to proteins, and to lipids by free radicals; (iii) role of antioxidants (e.g. glutathione) in the maintenance of cellular "redox homeostasis"; (iv) overview of ROS-induced signaling pathways; (v) role of ROS in redox regulation of normal physiological functions, as well as (vi) role of ROS in pathophysiological implications of altered redox regulation (human diseases and ageing). Attention is focussed on the ROS/RNS-linked pathogenesis of cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid arthritis, and ageing. Topics of current debate are also reviewed such as the question whether excessive formation of free radicals is a primary cause or a downstream consequence of tissue injury.

2.Sugino, N
Reprod Med Biol. 2005; 4: 31 – 44
Cells living under aerobic conditions always face oxygen paradox. Oxygen is necessary     for cells to maintain their lives. However, reactive oxygen species such as superoxide  radical ( inline image), hydroxyl radical (OH−) and hydrogen peroxide (H2O2) are generated from oxygen and damage cells. Oxidative stress occurs as a consequence of     excessive production of reactive oxygen species and impaired antioxidant defense systems. Antioxidant enzymes include: superoxide dismutase (SOD), which is a specific enzyme to scavenge superoxide radicals; copper-zinc SOD, located in the cytosol; and  manganese SOD, located in the mitochondria. Both types of SOD belong to the first enzymatic step to scavenge superoxide radicals.
It has been reported that a number of local factors such as cytokines, growth factors and eicosanoids are involved in the regulation of ovarian function, in addition to     gonadotropins and ovarian steroid hormones. Since reactive oxygen species are generated and SOD is expressed in the ovary, there is a possibility that reactive oxygen species and SOD work as local regulators of ovarian function. The present review reports that reactive oxygen species and their scavenging systems play important roles in several processes of reproductive physiology, including follicular development, oocyte maturation, ovulation, corpus luteum function and follicular atresia.

3. Espey, LL
Biol Reprod. 1980;22(1):73 – 106
Resumen no disponible

4. Nakamura, Y
Fertil Steril. 2003;80:1012 - 6.
OBJECTIVE:
To study the role of follicular melatonin on steroid production by human preovulatory follicles.
DESIGN:
In vivo comparative and in vitro culture studies.
SETTING:
University hospital.
PATIENT(S):
Thirty-six patients with tubal and/or male, but not endocrinological, infertility factors.
INTERVENTION(S):
Follicular fluid collection during IVF. In vitro granulosa cell culture from luteinizing or growing Graafian follicles.
MAIN OUTCOME MEASURE(S):
Follicular melatonin, P, T, and E(2) concentrations. In vitro P production by granulosa cells.
RESULT(S):
There was a positive correlation between follicular melatonin and P concentrations. Melatonin did not stimulate in vitro P production by granulosa cells from luteinizing or growing follicles. Melatonin, P, and E(2) concentrations were significantly higher, but T concentrations were lower, in large follicles than in small follicles.
CONCLUSION(S):
Preovulatory follicles contain a high amount of melatonin compared with that in small immature follicles; melatonin may play an important, but indirect, role in P production by human granulosa cells

5. Paszkowski T
Clin Chim Acta. 1995;236:173 - 80.
The purpose of this study was to evaluate the follicular fluid selenium status of in vitro fertilization patients in relation to their individual characteristics and treatment outcome. A total of 135 samples of follicular fluid were collected from 112 patients during transvaginal oocyte retrieval. Selenium levels and glutathione peroxidase (GSHPx) activity were measured in follicular fluid (FF) and blood serum. A selenium dependent GSHPx activity in follicular fluid has been demonstrated. Patients with unexplained infertility had significantly decreased follicular selenium levels as compared with those with tubal infertility or male factor. The mean GSHPx activity in follicles yielding oocytes which were subsequently fertilized exceeded that of the follicles with non-fertilized oocytes. Tobacco smoking resulted in significantly diminished follicular GSHPx activity. The Se/GSHPx pattern of the FF seems to be significant for the evaluation of the follicular milieu.

6. Acuña-Castroviejo, D
Cell Mol Life Sci. 2014;71:2997- 3025.
Endogenous melatonin is synthesized from tryptophan via 5-hydroxytryptamine. It is considered an indoleamine from a biochemical point of view because the melatonin molecule contains a substituted indolic ring with an amino group. The circadian production of melatonin by the pineal gland explains its chronobiotic influence on organismal activity, including the endocrine and non-endocrine rhythms. Other functions of melatonin, including its antioxidant and anti-inflammatory properties, its genomic effects, and its capacity to modulate mitochondrial homeostasis, are linked to the redox status of cells and tissues. With the aid of specific melatonin antibodies, the presence of melatonin has been detected in multiple extrapineal tissues including the brain, retina, lens, cochlea, Harderian gland, airway epithelium, skin, gastrointestinal tract, liver, kidney, thyroid, pancreas, thymus, spleen, immune system cells, carotid body, reproductive tract, and endothelial cells. In most of these tissues, the melatonin-synthesizing enzymes have been identified. Melatonin is present in essentially all biological fluids including cerebrospinal fluid, saliva, bile, synovial fluid, amniotic fluid, and breast milk. In several of these fluids, melatonin concentrations exceed those in the blood. The importance of the continual availability of melatonin at the cellular level is important for its physiological regulation of cell homeostasis, and may be relevant to its therapeutic applications. Because of this, it is essential to compile information related to its peripheral production and regulation of this ubiquitously acting indoleamine. Thus, this review emphasizes the presence of melatonin in extrapineal organs, tissues, and fluids of mammals including humans.

7. Tamura, H
J Pineal Res 2008;44:280 - 7.
We investigated the relationship between oxidative stress and poor oocyte quality and whether the antioxidant melatonin improves oocyte quality. Follicular fluid was sampled at oocyte retrieval during in vitro fertilization and embryo transfer (IVF-ET). Intrafollicular concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in women with high rates of degenerate oocytes were significantly higher than those with low rates of degenerate oocytes. As there was a negative correlation between intrafollicular concentrations of 8-OHdG and melatonin, 18 patients undergoing IVF-ET were given melatonin (3 mg/day), vitamin E (600 mg/day) or both melatonin and vitamin E. Intrafollicular concentrations of 8-OHdG and hexanoyl-lysine adduct were significantly reduced by these antioxidant treatments. One hundred and fifteen patients who failed to become pregnant with a low fertilization rate (< or =50%) in the previous IVF-ET cycle were divided into two groups during the next IVF-ET procedure; 56 patients with melatonin treatment (3 mg/day) and 59 patients without melatonin treatment. The fertilization rate was improved by melatonin treatment compared to the previous IVF-ET cycle. However, the fertilization rate was not significantly changed without melatonin treatment. Oocytes recovered from preovulatory follicles in mice were incubated with H2O2 for 12 hr. The percentage of mature oocytes with a first polar body was significantly reduced by addition of H2O2 (300 microm). The inhibitory effect of H2O2 was significantly blocked by simultaneous addition of melatonin. In conclusion, oxidative stress causes toxic effects on oocyte maturation and melatonin protects oocytes from oxidative stress. Melatonin is likely to improve oocyte quality and fertilization rates.

8. Carroll, J
Rev Reprod. 1996;1:137 - 43
Oogenesis involves the production of an oocyte that can undergo fertilization and support early development. The stimulus that initiates embryogenesis is an increase in the concentration of intracellular Ca2+ in the cytoplasm of the oocyte at the time of fertilization. The development of the ability of the oocyte to release Ca2+ in response to the fertilizing spermatozoon is an essential step in the process of oogenesis. Mammalian oocytes are particularly useful for studying the development of Ca2+ signalling systems, owing to the series of Ca2+ oscillations generated at fertilization, compared with the monotonic Ca2+ increase seen in nonmammalian species. Recent evidence has revealed that Ca2+ release mechanisms are modified during oogenesis. The maximal sensitivity of Ca2+ release is reached in the final stages of oocyte maturation, just before the optimal time for fertilization. In this review, we consider the mechanism underlying Ca2+ release in mammalian oocytes and discuss how the release mechanisms are modified during oocyte maturation. The tight co-ordination of the differentiation of the Ca2+ signalling system with the development of the oocyte provides a means of ensuring successful activation at the time of fertilization. Finally, we consider the consequences for embryo development in circumstances in which the co-ordination is lost.

9. Chiu, TT
Hum Reprod, 2002; 17: 1591 - 6
BACKGROUND:
The follicular microenvironment is an important determinant of oocyte development. The aim of this study was to examine whether the myo-inositol (MI) content in human follicular fluid (FF) was associated with better oocyte quality.
METHODS:
A total of 53 patients treated with IVF was recruited to a prospective observational study. FF and serum samples collected were divided into two groups: group A consisted of FF associated with matured and fertilized oocytes, whilst group B was from follicles with immature and unfertilized oocytes.
RESULTS:
Patient's age, total ampoules of HMG used, days of stimulation, basal levels of FSH, estradiol (E(2)) levels on the day of HCG, and serum MI content were not significantly different between the two groups. FF volume and its MI content were significantly higher in group A compared with group B (P < 0.05). The levels of MI in FF were positively correlated with the amount of E(2) in their corresponding FF samples and also correlated with embryo quality.
CONCLUSIONS:
We propose that higher concentrations of MI and E(2) in human FF appear to play a role in follicular maturity and provide a marker of good quality oocytes.

10. Pesty, A
Mol Reprod Dev, 1994. 38: 187 - 99
The incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose-dependent and reversible manner. When myo-inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP3) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo-inositol nor InsP3 were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP3-independent and seems to be influenced negatively via underdevelopment of microtubular structures.

11. Pacchiarotti, A
Gynecol Endocrinol 2016; 32:69 - 73
Polycystic ovarian syndrome (PCOS) induces anovulation in women of reproductive age, and is one of the pathological factors involved in the failure of in vitro fertilization (IVF). Indeed, PCOS women are characterized by poor quality oocytes. Therefore, a treatment for enhancing oocyte quality becomes crucial for these patients. Myo-Inositol and melatonin proved to be efficient predictors for positive IVF outcomes, correlating with high oocyte quality. We tested the synergistic effect of myo-inositol and melatonin in IVF protocols with PCOS patients in a randomized, controlled, double-blind trial. Five-hundred twenty-six PCOS women were divided into three groups: Controls (only folic acid: 400 mcg), Group A (Inofolic® plus, a daily dose of myo-inositol: 4000 mg, folic acid: 400 mcg, and melatonin: 3 mg), and Group B (Inofolic®, a daily dose of myo-inositol: 4000 mg, and folic acid: 400 mcg). The main outcome measures were oocyte and embryo quality, clinical pregnancy and implantation rates. The treatment lasted from the first day of the cycle until 14 days after embryo transfer. Myo-inositol and melatonin have shown to enhance, synergistically, oocyte and embryo quality. In consideration of the beneficial effect observed in our trial and on the bases of previous studies, we decided to integrate routinely MI and M supplementation in the IVF protocols. The same treatment should be taken carefully in consideration in all procedures of this kind.

12. Unfer, V
Gynecol Endocrinol, 2011. 27:857 - 61
BACKGROUND:
Several factors can affect oocyte quality and therefore pregnancy outcome in assisted reproductive technology (ART) cycles. Recently, a number of studies have shown that the presence of several compounds in the follicular fluid positively correlates with oocyte quality and maturation (i.e., myo-inositol and melatonin).
AIM:
In the present study, we aim to evaluate the pregnancy outcomes after the administration of myo-inositol combined with melatonin in women who failed to conceive in previous in vitro fertilization (IVF) cycles due to poor oocyte quality.
MATERIALS AND METHODS:
Forty-six women were treated with 4 g/day myo-inositol and 3 mg/day melatonin (inofolic® and inofolic® Plus, Lo.Lipharma, Rome) for 3 months and then underwent a new IVF cycle.
RESULTS:
After treatment, the number of mature oocytes, the fertilization rate, the number of both, total and top-quality embryos transferred were statistically higher compared to the previous IVF cycle, while there was no difference in the number of retrieved oocyte. After treatment, a total of 13 pregnancies occurred, 9 of them were confirmed echographically; four evolved in spontaneous abortion.
CONCLUSION:
The treatment with myo-inositol and melatonin improves ovarian stimulation protocols and pregnancy outcomes in infertile women with poor oocyte quality.