European Review for Medical and Pharmacological Sciences 2011; 15: 509-514 Effects of Myo-Inositol supplementation on oocyte’s quality in PCOS patients: a double blind trial

L. CIOTTA, M. STRACQUADANIO, I. PAGANO, A. CARBONARO, M. PALUMBO, F. GULINO

Microbiological and Gynecological Science Department, Gynecology Section “Santo Bambino” Hospital (Catania), University of Catania (Italy)

Abstract. – Background: Polycystic ovary Introduction syndrome is the most common cause of chronic anovulation infertility in women in fertile period, and it’s characterized by an increased produc- Polycystic Ovary Syndrome (PCOS) is a com- tion of androgens and estrogens. The adminis- plex disease characterized by various endocrine tration of D-chiro-inositol, a B complex vitamin, disorders that can be the potential cause of was associated with a decreased of serum anovulation and hyperandrogenism condition. testosterone and simultaneously, due to its abili- This heterogenous syndrome affects about 5- ty to increase insulin sensitivity, women who re- 10% of female population in the reproductive ceived D-chiro-inositol showed a great improve- age, and it can be considered as the most com- ment of the ovulary function. Besides, the sup- plementation of inositol improves the oocytes’ mon endocrine disorder affecting women during 1 quality and increase the number of oocytes col- the reproductive life . lected after ovarian stimulation in patients un- PCOS cannot be merely considered a local dergoing IVF (in vitro fertilization). ovarian dysfunction or a central hypothalamus- Aim: The aim of this study is to determine the ovary-pituitary defect, but it is the expression of effects of myo-inositol on oocyte’s quality on a a complex functional alteration of the whole re- sample of women with polycystic ovary syndrome. Material and Methods: The patients were di- productive system. vided into two groups: patients of Group A in- Under a hormonal point of view, the micropoly- took 2 g of myo-inositol + 200 µg of folic acid (In- cystic ovary is characterized by an increased pro- ofolic®, LO.LI. Pharma, Rome, Italy) while Group duction of androgens and estrogens, and a dissoci- B only 200 µg of folic acid, both groups took the ation of gonadotropins serum concentrations: ele- treatment twice a day, continuously for 3 vated luteinizing hormone (LH), low or normal months. follicle stimulating hormone (FSH) and LH/FSH Results: At the end of treatment, the number of follicles of diameter >15 mm, visible at ultra- ratio that usually exceeds 2.5 in the typical forms. sound during stimulation, and the number of In the blood of PCOS patients testosterone oocytes recovered at the time of pick-ups were (T), androstenedione (AS), dehydroepiandros- found to be significantly greater in the group terone (DHEA), DHEA-S (sulfate), 17-hydrox- treated with myo-inositol, so as the aver-age yprogesterone (17-OHP) and estrone resulted el- number of embryos transferred and embryo evated. The circulating levels of sex hormone- Score S1. Significantly reduced was the average number of immature oocytes (vesicles germ and binding globulin (SHBG) are instead lower. degenerated oocytes) too. The enhancing peripheral conversion of an- Conclusions: These data suggest that myo- drostenedione to estrone leads to the modest rela- inositol may be useful in the treatment of PCOS tive hyperandrogenism. patients undergoing ovulation induction, both SHBG levels are reduced of about 50% due to for its insulin-sensitizing activity, and its role in the increased levels of insulin2-4. oocyte maturation. The syndrome’s etiology is still unknown, but it Key Words: is probably multifactorial, due to an excessive E1 production, or to an alteration of the primitive hy- Inositol, Oocyte’s quality, Polycystic ovary syn- In vitro pothalamic regulation and of the ovarian and/or drome, Infertility, fertilization. adrenal steroidogenesis.

Corresponding Author: Mariagrazia Stracquadanio, MD; e-mail: [email protected] 509 L. Ciotta, M. Stracquadanio, I. Pagano, A. Carbonaro, M. Palumbo, F. Gulino

The diagnosis of PCOS is based on the clini- Clomiphene is a drug normally used for this cal, hormonal and ultrasound patterns. In accor- purpose: it is a weak estrogen that acts also as dance with the Rotterdam Criteria, drawn in anti-estrogen. Probably, it interacts with the hy- 2003, PCOS diagnosis can be made only after pothalamic estrogen receptors, displacing the en- the exclusion of other causes of hyperandro- dogenous estradiol and creating a condition of genism and amenorrhea, and in the presence of at artificial hypoestrogenism, due to its biological least 2 of the following criteria: activity almost absent in this district. Hypothala- mic centers, responsible for gonadotropin-releas- • Oligo- and/or anovulation with menstrual ir- ing hormone (GnRH) release are thus stimulated regularities; to greater activity. Following the administration • Elevated levels of circulating androgens or of clomiphene, in fact, the frequency of pulsatile clinical manifestation of hyperandrogenism; secretion of LH and FSH increases, while the • Transvaginal pelvic ultrasound evidence of amplitude remains unchanged. Ovulation in micropolycystic ovary. PCOS is induced in 80% of cases, while preg- nancy occurs in 20% of cases. Due to the pulsatility of LH, only one blood Where no response to the treatment with parameter is not enough for the PCOS diagnosis, clomiphene and metformin was obtained, or and there is no unanimous consensus on which where an in vitro fertilization/intracytoplasmic androgen blood’s level should be considered for sperm injection (IVF/ICSI) was necessary, ovula- a precise diagnosis (total or free testosterone, tion induction was performed by the administra- testosterone/SHBG ratio or androstenedione). tion of gonadotropins. Gonadotropins used for Usually, elevated levels of only DHEA or 17- this purpose are obtained from the urine of post- OHP may exclude the diagnosis of PCOS. menopausal women (Menotrophin). Recently, Since menarche, or after a short period, men- gonadotropins obtained with biosynthetic tech- strual cycles show an irregular rhythm. In many nique from recombinant DNA have been intro- cases they gradually distance themselves from duced (Follicotropin α and Follicotropin β). The each other, up to result in short periods of amen- goal of the therapy with gonadotropins, or rather orrhea or in permanent amenorrhea. Menstrual with FSH, is acting on the follicles in the last dysfunction in women affected by PCOS may stage of their maturation process that, under manifest in different ways, but the probably most physiological conditions is restricted to the first common way is anovulation with erratic bleed- two weeks of the menstrual cycle in which ovula- ings. tion occurs5,7. Androgens excess is responsible for hirsutism, The aim of this study is to determine the ef- oily skin, acne and, in the ovary, for the thicken- fects of Myo-Inositol, a compound belonging to ing of the tunica albuginea. The degree of hir- vitamin B complex, on oocyte’s quality in a sutism can be measured with the Ferriman-Gall- group of patients with PCOS, suffering from wey score. chronic anovulation and infertility, undergoing In rare cases virilization patterns can be ob- medically assisted reproduction techniques served, with increased size of clitoris, muscle (ART), such as IVF and ICSI. mass hypertrophy, deep voice, temporal balding Scientific studies have shown that D-chiro-in- and masculine aspect. In these cases, however, a ositol, thanks to its ability to increase insulin sen- lower ovarian or an adrenal androgen-secreting sitivity, has beneficial effects on ovulation and on neoplasia must be excluded. the androgens production in women with PCOS. At the same time an overweight pattern, up to The administration of D-chiro-inositol was asso- obesity can be associated to the syndrome. ciated with a decreased of serum testosterone8 PCOS is one of the most common endocrine and increased of SHBG concentration. Simulta- causes of female infertility: if you want to get neously to the reduction of insulin secretion, pregnant, ovulation should be induced5. women who received D-chiro-inositol showed a Ovulatory cycles are obtained, usually, after great improvement of the ovulary function9,10. the overweight correction, or immediately after Scientific evidence has also shown that the sup- the estrogen-progestins suspension. If it does not plementation with inositol contributes to reduc- happen, ovulation should be induced pharmaco- ing the amount of FSH necessary to ovulation, to logically (usually associated with metformin ad- improving oocyte’s quality (reduction of the total ministration)6. amount of the germinal vesicles and the degener-

510 Effects of Myo-Inositol supplementation on oocyte’s quality in PCOS patients: a double blind trial ated oocytes) and to increasing the number of Oocyte’s quality assessment was performed oocytes collected after ovarian stimulation in pa- after the oocyte pick-up conducted during the as- tients undergoing ART techniques, as IVF or IC- sisted reproductive technology procedure in SI11-13. Inositol cannot be defined exactly as a vit- which patients have been submitted. The ICSI or amin, but it is considered a vitamin factor be- IVF includes several phases (ovarian stimulation, longing to B complex. In the human organism it oocyte collection, oocyte quality assessment, is present in the phospholipids, and it can stimu- oocytes in-vitro fertilization, embryo culture and late endogenous production of lecithin. Its role scoring, embryo transfer), all crucial for the suc- also includes a specific biological activity of con- cess of the technique. They were all followed by trol on fat and sugar metabolism, and on the cel- the medical team of the Reproduction Pathophys- lular function of the nervous system. It is also es- iology Centre of “Santo Bambino” Hospital, in sential to hair growth and it can prevent baldness. Catania at the IVF clinic with the attached Scientific studies revealed that diabetic subjects surgery room. eliminate amounts of inositol significantly higher that no-diabetic ones14. Statistical Analysis In case of insulin resistance or type II diabetes, The comparison between Group A and Group inositol helps to improve the whole clinical pat- B was performed using: tern. In these cases, inositol may be useful to pre- vent and to correct pathophysiological mecha- • Χ2 test for qualitative data (β-hCG positivity); nisms underlying the metabolic and reproductive • Student t test for quantitative data normally abnormalities related to PCOS15-18. distributed (age, BMI, total FSH units admin- istered, number of follicles of diameter >15 mm); • U test for quantitative data not normally dis-

Materials and Methods tributed (days of stimulation, E2 maximum peak, number of oocytes retrieved). All the patients were enrolled and treated in the Department of Gynecological Sciences (“Santo Bambino” Hospital, Catania), at the Gy- necological Endocrinology Clinics and Human Results Reproduction Pathophysiology Centre. In the 12- month enrollment phase a total of 34 women, During the study period, patients were ran- aged <40 years with polycystic ovary syndrome domly divided into two groups, as described be- (PCOS) were selected. fore, and the investigation was performed in a PCOS diagnosis was indicated by oligo-amen- double-blind design. orrhea (six or fewer menstrual cycles during a No significant differences were found between period of one year), hyperandrogenism (hir- the two groups in mean age and body mass index sutism, acne or alopecia) or hyperandrogenemia (BMI). (elevated levels of total or free testosterone), and Total r-FSH units administered for the ovari- typical feature of ovaries at ultrasound scan. an stimulation were significantly reduced in Concomitant endocrine and metabolic patholo- group A. gies, as hypothyroidism, hyperthyroidism, diabetes As reported in literature, peak E2 levels at hu- mellitus, androgen-secreting cancers, adrenal hy- man chorionic gonadotropin (hCG) administra- perplasia, Cushing syndrome were excluded. tion were lower in group A, but our data were not The ICSI or IVF procedures were recommend- statistically significant. ed after the evaluation of the sperm semen of the Two cycles were cancelled in group A, where- male partner. as in group B five cycles were suspended, be-

According to a randomization table, patients cause of peak E2 >4,000 pg/mL (risk of hyper- were divided into two groups: patients of Group A stimulation). intook 2 g of myo-inositol + 200 µg of folic acid The number of follicles with a diameter >15 (Inofolic®, LO.LI. Pharma, Rome, Italy) while mm, visible at ultrasound scan during stimula- Group B only 200 µg of folic acid, both groups tion, and the number of oocytes retrieved at the took the treatment twice a day, continuously for 3 pick-up resulted significantly higher in the myo- months. inositol-treated group (Table I).

511 L. Ciotta, M. Stracquadanio, I. Pagano, A. Carbonaro, M. Palumbo, F. Gulino

Table I. Retrieved oocytes at the pick-up. therapies in spontaneous ovulation restoration, with a potential positive action even on meiotic Percentiles oocyte maturation. These therapies seem to di- Group Median 25° 75° rectly influence steroidogenesis, by reducing an- drogen production in theca cells. In fact, the ad- A121016ministration of D-chiro-inositol has been demon- B 8.50 6.25 10.75 strated to increase the insulin action in PCOS pa- 10 P < 0.05 tients, improving ovulatory function , and reduc- ing serum testosterone concentration8, 9,19. Nowadays, there are few data on the action and effects of myo-inositol, a precursor of D-chi- The mean number of immature oocytes (ger- ro-inositol, on the anovulatory women in repro- minal vesicles and degenerated oocytes) was sig- ductive age or on the spontaneous ovulation in nificantly reduced, and there was an increasing stimulated cycles. trend of the rate of oocytes in metaphase II Anyway, myo-inositol is an important con- (MII), that are oocytes characterized by not visi- stituent of the follicular microenvironment, play- ble germinal vesicles and visible first polar body ing a key role in the nuclear and cytoplasmic (Table II). oocyte’s development. No statistical significance in the number of In the assisted reproduction techniques, in fertilized embryos was emerged, but in group A fact, the supplementation with myo-inositol is the mean number of transferred embryos resulted positively related to meiotic progression of significantly higher, with higher amounts of mouse germinal vesicle oocytes, enhancing in- score 1 embryos in comparison with lower-quali- tracellular Ca2+ oscillation20. Furthermore, high- ty embryos (Table III). er concentrations of myo-inositol in human fol- In compliance with the italian ART law, no licular fluid provide a marker of good-quality more than three embryos were transferred. No oocytes21. differences in the total number of biochemical Our study is one of the fews focusing on this pregnancies were detected. molecule, that belongs to vitamin B complex, and on its effects in PCOS patients undergoing ovulation induction. Preliminary data in our hands show that, in PCOS patients the treatment Discussion with myo-inositol and folic acid, compared with folic acid alone, reduces the number of germinal Polycystic ovary syndrome (PCOS) is one of vesicles and degenerated oocytes, without com- the most common female endocrine disorders. promising the total number of retrieved oocytes. Insulin-resistance and hyperinsulinemia are These results, as other trials’, suggest that myo- strictly correlated with the phenotype of a large inositol has a positive effect on mature oocytes part of PCOS women. development22. A defect in the insulin’s action has been sus- Furthermore, it is well known that ovulation pected, probably because of a deficiency of D- induction in PCOS patients is a pivotal matter, chiro-inositol, that is a component of inositol even because of the risk of the ovarian hyper- phosphoglycans. Insulin-lowering drugs, particu- stimulation syndrome23-24. Elevated basal serum larly different forms of inositol, represent novel levels of androgens are involved in the produc-

Table II. Metaphase II (MII) oocytes, degenerated oocytes and germinal vesicles.

Group A Group B

Frequency % Frequency % P

MII oocytes 176 82.24 160 66.87 NS Degenerated oocytes 2 0.93 23 14.37 0.02 Germinal vesicles 3 1.4 15 9.37 0.02

512 Effects of Myo-Inositol supplementation on oocyte’s quality in PCOS patients: a double blind trial

Table III. Number of score 1 embryos.

Group A Group B

Frequency % Frequency % P

Score 1 embryos 30 68.1 9 29 < 0.01

6) ACOG COMMITTEE ON PRACTICE BULLETINS. Polycystic tion of high serum E2 levels, as typically detected in PCOS patients undergoing ovulation induction ovary syndrome. Int J Gynecol Obstet 2003; 80: with exogenous gonadotropins. 335-348. Because myo-inositol is a D-chiro-inositol 7) TOSCANO V. S INDROME DELL’OVAIO POLICISTICO. IN: AN- DREANI D, TAMBURANO G. Manuale di Terapia delle precursor, an insulin-sensitizing action on the Malattie Endocrine e Metaboliche. Delfino Anto- ovary may be similarly hypothesized, with a sub- nio Editore, 2000. sequent positive action on the hormonal profile, 8) COSTANTINO D, MINOZZI G, MINOZZI F, G UARALDI C. particularly on basal serum testosterone reduc- Metabolic and hormonal effects of myo-inositol in tion8,9,25. In fact, in patients treated with myo-in- women with polycystic ovary syndrome: a double- ositol plus exogenous gonadotropins a significant blind trial. Eur Rev Med Pharmacol Sci 2009; 13: 105-110. reduction in E2 levels at hCG administration was found. As consequence, it can be supposed that 9) NESTLER JE, JAKUBOWICZ DJ, REAMER P, G UNN RD, AL- this protocol could be adopted to reduce the risk LAN G. Ovulatory and metabolic effects of D chiro- inositol in the polycystic ovary syndrome. N Engl of hyperstimulation in such patients. J Med 1999; 340: 1314-1320. In conclusion, these observations suggest that 10) GERLI S, PAPALEO E, FERRARI A, DI RENZO GC. Ran- myo-inositol may be useful in the treatment of domized, double blind placebo-controlled trial: PCOS patients undergoing ovulation induction, effects of Myo-inositol on ovarian function and both for its insulin-sensitizing activity, and its metabolic factors in women with PCOS. Eur Rev role in oocyte maturation. Med Pharmacol Sci 2007; 11: 347-354. 11) PAPALEO E, UNFER V, B AILLARGEON JP , FUSI F, O CCHI F, DE SANTIS L. Myo-inositol may improve oocyte quality in intracytoplasmic sperm injection cycles. A prospective, controlled, randomized trial. Fertil References Steril 2009; 91: 1750-1754. 12) PAPALEO E, UNFER V, B AILLARGEON JP, DE SANTIS L, FUSI 1) SATTAR N, HOPKINSON Z, GREER IA. Insulin-sensiting agents in polycystic ovary syndrome. Lancet F, B RIGANTE C, MARELLI G, CINO I, REDAELLI A, FERRARI 1998; 351: 305. A. Myo-inositol in patients with polycystic ovary syndrome: a novel method for ovulation induction. 2) BAPTISTE CG, BATTISTA MC, TROTTIER A, BAILLARGEON JP. Gynecol Endocrinol 2007; 23: 700-703. Insulin and hyperandrogenism in women with 13) CHIU TT, ROGERS MS, LAW EL, BRITON-JONES CM, CHE- polycystic ovary syndrome. J Steroid Biochem UNG LP, HAINES CJ. Follicular fluid and serum con- Mol Biol 2010; 122: 42-52. centrations of myo-inositol in patients undergoing 3) FLAMIGNI C, VENTUROLI S, PORCU E. La sindrome del- IVF: relationship with oocyte quality. Hum Reprod l’ovaio micropolicistico; Considerazioni cliniche ed 2002; 17: 1591-1596. eziopatogenetiche. In: Genazzani, Volpe: En- 14) KNOWLER WC, BARRETT-CONNOR E, FOWLER SE, HAM- docrinologia Ginecologica. Fisiopatologia, Clinica MAN RF, LACHIN JM, WALKER EA, NATHAN DM; DIA- e Strategie Terapeutiche. Monduzzi Editore, BETES PREVENTION PROGRAM RESEARCH GROUP. Reduc- 1984. tion in the incidence of type 2 diabetes with 4) FILICORI M, FLAMIGNI C, CAMPANIELLO E, MERIGGIOLA lifestyle intervention or metformin. N Engl J Med MC, MICHELACCI L, VALDISERRI A, FERRARI P. Polycystic 2002; 346: 393-403. ovary syndrome: abnormalities and management 15) LOWENSTEIN EJ. Diagnosis and management of the with pulsatile gonadotropin-releasing hormone and dermatologic manifestations of the polycystic ovary gonadotropin-releasing hormone analogs. Am J syndrome. Dermatol Ther 2006; 19: 210-223. Obstet Gynecol 1990; 163: 1737-1742. 16) AZZIZ R, SANCHEZ LA, KNOCHENHAUER ES, MORAN C, 5) AROSIO M, PERSANI L, FAGLIA G. Sindrome dell’ovaio LAZENBY J, STEPHENS KC, TAYLOR K, BOOTS LR. Andro- policistico; Malattie endocrine dell’ovaio. In: Faglia gen excess in women: Experience with over 1000 G, Beck-Peccoz P. Malattie del sistema endocrino consecutive patients. J Clin Endocrinol Metab e del metabolismo. McGraw-Hill, 2006. 2004; 89: 453-462.

513 L. Ciotta, M. Stracquadanio, I. Pagano, A. Carbonaro, M. Palumbo, F. Gulino

17) AMERICAN DIABETES ASSOCIATION. Position statement: IVF: relationship with oocyte quality. Hum Reprod Gestational diabetes mellitus. Diabetes Care 2002; 17: 1591-1596. 2004; 7(Suppl. 1): S88-S90. 22) GOUD PT, GOUD AP, VAN OOSTVELDT P, D HONT M. 18) JOINT NATIONAL COMMITTEE ON PREVENTION, DETEC- Presence and dynamic redistribution of type I in- TION, EVALUATION, AND TREATMENT OF HIGH BLOOD ositol 1,4,5-trisphosphate receptors in human PRESSURE. The sixth report of the Joint National oocytes and embryos during in-vitro maturation, Committee on Prevention, Detection, Evaluation, fertilization and early cleavage divisions. Mol Hum and Treatment of High Blood Pressure. Arch Int Reprod 1999; 5: 441-451. Med 1997; 157: 2413-2446. 23) TUMMON I, GAVRILOVA-JORDAN L, ALLEMAND MC, SES- 19) BAILLARGEON JP, DIAMANTI-KANDARAKIS E, OSTLUND RE SION D. Polycistic ovaries and ovarian hyperstimu- JR, APRIDONIDZE T, I UORNO MJ, NESTLER JE. Altered D- lation syndrome: a systematic review. Acta Obstet chiro-inositol urinary clearance in women with Gynecol Scand 2005; 84: 611-616. polycystic ovary syndrome. Diabetes Care 2006; 24) BATTAGLIA C, MANCINI F, P ERSICO N, ZACCARIA V, DE 29: 300-305. ALOYSIO D. Ultrasound evaluation of PCO, PCOS 20) CHIU TT, ROGERS MS, BRITON JONES CM, HAINES C. Ef- and OHSS. Reprod Biomed Online 2004; 9: 614- fect of myo-inositol on the in-vitro maturation and 619. subsequent development of mouse oocytes. Hum 25) IUORNO MJ, JAKUBOWICZ DJ, BAILLARGEON JP, DILLON Reprod 2003; 18: 408-416. P, G UNN RD, ALLAN G, NESTLER JE. Effects of d- 21) CHIU TT, ROGERS MS, LAW EL, BRITON-JONES CM, CHE- chiro-inositol in lean women with the polycystic UNG LP, HAINES CJ. Follicular fluid and serum con- ovary syndrome. Endocr Pract 2002; 8: 417- centrations of myo-inositol in patients undergoing 423.

514