High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries

Abstract

Administration of 1000-1500 mg/day D-Chiro-Inositol (DCIns) or a combination of Myo-Inositol (MyoIns) and DCIns in their plasma molar ratio (40:1) for three or more months are among recommended treatments for metabolic syndrome and/or Polycystic Ovary Syndrome (PCOS). We previously confirmed the efficacy of this formulation (8.2 mg/day MyoIns and 0.2 mg/day DCIns for 10 days) in a mouse PCOS model, but also observed negative effects on ovarian histology and function of formulations containing 0.4-1.6 mg/day DCIns. We therefore analyzed effects of higher doses of DCIns, 5, 10 and 20 mg/day, administered to young adult female mice for 21 days, on ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. Five mg/day DCIns (human correspondence: 1200 mg/day) altered ovarian histology, increased serum testosterone levels and reduced the amount of aromatase of negative controls, suggesting the induction of an androgenic PCOS model. In contrast, 10-20 mg/day DCIns (human correspondence: 2400-4800 mg/day) produced ovarian lesions resembling those typical of aged mice, and reduced serum testosterone levels without affecting aromatase amounts, suggesting a failure in steroidogenic gonadal activity. Notwithstanding physiological/biochemical differences between mice and humans, the observed pictures of toxicity for ovarian histology and function recommend caution when administering DCIns to PCOS patients at high doses and/or for periods spanning several ovulatory cycles.

Keywords: PCOS model; androgenic phenotype; aromatase; inositol; letrozole; menopause; mouse ovary; testosterone.

Figure 1

Increase in mouse weights during the 21-day treatment. Histograms represent percent weight increases (mean ± SD) depending on the molecules and doses received. One Way ANOVA, p < 0.005; *, p < 0.05 versus H₂O and letrozole; **, p < 0.01 versus DCIns 1000.

Figure 2

Macroscopic view of mouse uterus-ovary complexes at the end of the 21 day-treatment. Gross morphology of typical uteri and ovaries from mice that received, from left to right: (A) plain water (negative control), (B) DCIns 250, (C) letrozole (positive control), (D) DCIns 500 and (E) DCIns 1000. Note the longer extension and thicker appearance of a typical uterus from control mice (A) compared with the shorter and thinner appearance of the uteri from DCIns- and letrozole-administered mice (B–E). All uteri are shown at the same scale (mm).

Figure 3

Ovarian histology of mice at the end of the 21 day-treatment. (A1,A2) Ovarian sections from mice that received plain water (negative control), showing primary, secondary, and tertiary follicles as well as a corpus luteum (A1). These features are typical of normally cycling mice. (B1,B2) Sections from mice subjected to 21-day treatment with DCIns 250, showing primary, secondary, tertiary and cystic follicles devoid of oocytes. These features resemble typical signs of PCOS. (C1,C2) Sections from mice subjected to 21-day treatment with letrozole (positive controls), showing paucity of follicles and a large cyst, typically modeling human PCOS. (D1,D2) Sections from mice subjected to 21-day treatment with DCIns 500, showing secondary and tertiary follicles as well as follicles with signs of hyperproliferation and extension of the stromal compartment. (E1,E2) Sections from mice subjected to 21-day treatment with DCIns 1000, showing paucity of secondary and tertiary follicles as well as large follicles with signs of hyperproliferation and extension of the stromal compartment. Hematoxylin-eosin. Scale bars, 100 μm.

Figure 4

Histological features of mouse ovaries at the end of the 21 day-treatment. (A,B) Ovarian sections from mice that received plain water (negative control), showing a primary and a tertiary follicle (A) and a corpus luteum (B). (C) A section from a DCIns 250-treated mouse with cysts. (D) A section from a letrozole-treated mouse (positive control) with large cysts. (E) A section from a DCIns 500-treated mouse with follicular hyperproliferation. (F) A section from a DCIns 1000-treated mouse with stromal extension. Hematoxylin-eosin. Scale bars, 100 μm.

Figure 5

Number of ovarian follicles at different stages and of different types at the end of the treatment. Histograms represent numbers of follicle of various types in abscissa (mean ± SD). *, difference in group composition vs. negative control ovaries; p < 0.05, calculated by repeated measures ANOVA.

Figure 6

Extension of theca and granulosa cell layers in ovarian follicles. (A) negative control mice; (B) mice subjected to 21-day treatment with DCIns 250; (C) mice subjected to 21-day treatment with letrozole; and (D) mice subjected to 21-day treatment with DCIns 500. The thickness of granulosa cell- (black bars) and theca cell-layers (white bars) is marked. Hematoxylin-eosin. Scale bars, 50 μm. Note the different aspect of oocytes in all panels.

Figure 7

Levels of aromatase in the ovaries of mice at the end of the 21-day treatment. (A) Representative Western blot of aromatase in protein extracts from the ovaries of mice under the experimental conditions indicated. (B) Densitometric analysis of aromatase/GAPDH. Values represent the mean value ± SD of extracts from three independent experiments. *, difference vs. negative control mice, p < 0.05.