P–618 D-Chiro-Inositol and mouse ovary: A new strange case of Dr Jekyll and Mr Hyde?

Study question Low doses of D-Chiro-Inositol are beneficial in the treatment of a PCOS mouse model. However, high doses are detrimental for ovarian histology/function. Is D-Chiro-Inositol toxic for the mammalian ovary? Summary answer Five mg/day D-Chiro-Inositol for 21 days produced PCOS-like histological/hormonal features. Ten/20 mg/day for 21 days induced ovarian/hormonal states resembling those typical of aged mice. What is known already Administration of Myo-Inositol and D-Chiro-Inositol combined according to their plasma molar ratio of 40:1 has beneficial effects in the management of human PCOS. We confirmed the efficacy of this formulation, containing 0.2 mg/day D-Chiro-Inositol, in a mouse model of PCOS. However, formulations containing higher amounts of DCIns had negative effects on ovarian histology and mouse fertility. We investigated possible ovarian toxicity of D-Chiro-Inositol, studying its effects after administration to 30-day-old female mice for 21 days. Young adult mice reproduced the condition of young women possibly facing reproductive/metabolic problems, such as PCOS. Study design, size, duration The effects of various doses of D-Chiro-Inositol were analysed on mouse ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. The 21-day period follows normal protocols of pharmacologic PCOS induction in the mouse and spans five ovulatory cycles. Doses employed, 5, 10, 20 mg/day, correspond to doses of 1200, 2400, 4800 mg/day in humans. The first dose is in the range of 1000–1500 mg/day currently prescribed to PCOS patients in clinical practice. Participants/materials, setting, methods Five mice/treatment were provided with water administering various doses of D-Chiro-Inositol or 0,5 mg/day letrozole as PCOS-positive controls, for 21 days. At the end of the period, ovulatory cycles were analysed by observations of vaginal cells after vaginal smears; ovarian histology was evaluated by sectioning, hematoxylin-eosin staining and light-transmission microscopy; serum testosterone levels were measured by ELISA; and expression of the ovarian enzyme aromatase was assayed by Western Blots. Main results and the role of chance The estrus cycle progressed normally in negative control mice, but was arrested at day 8–10 in the majority of mice under all pharmacologic treatments. Uteri of negative control mice displayed the typical aspect of mature and cycling animals. Uteri of all other mice had an immature/metestrus-diestrus-like aspect, typical of non-cycling animals. Ovaries of negative control mice showed a normal presence of primary, secondary and tertiary follicles containing a growing oocyte, and of corpora lutea. Ovaries from mice treated with 5 mg/day D-Chiro-Inositol or 0,5 mg/day letrozole had apparently normal primary and secondary follicles but also cystic tertiary follicles resembling those found in human PCOS. Ovaries from mice treated with 10 or 20 mg/day D-Chiro-Inositol had scarce primary and secondary follicles, a very limited number of tertiary follicles and no cystic follicles, but large follicles/areas with diffused cell proliferation. The typical ovarian structure was lost, especially in the highest dosage. Treatments with 5 mg/day D-Chiro-Inositol and 0,5 mg/day letrozole increased serum testosterone levels above those of negative control mice, but the former reduced, while the latter increased aromatase levels relative to negative controls. Other treatments had no apparent effects on either testosterone or aromatase levels. Our experimental paradigm makes the role of chance highly improbable. Limitations, reasons for caution The strength of our study relies on the use of an animal model representative of general human tissue organisation and physiological pathways. One weakness consists in the lack of data on serum estrogen levels, due to the paucity of blood provided by a single mouse and the ELISA sensitivity. Wider implications of the findings: Under all experimental conditions, D-Chiro-Inositol negatively affected ovarian histology and function. Notwithstanding physiological/biochemical differences between mice and humans, caution is therefore recommended when administering D-Chiro-Inositol to PCOS patients at doses corresponding to those we employed in the mouse and/or for long periods, since it may result ineffective or even toxic. Trial registration number Not applicable

Postembryonic developmental changes in ovarian histology and associated 17β-oestradiol and progesterone profiles in guinea hens (Numida meleagris)

There is a paucity of information on the reproductive biology of guinea hens. A study was conducted to document postembryonic developmental changes in the ovarian histology and associated progesterone and 17β-oestradiol profiles until 32 weeks of age (WOA). Highly significant increases (p<0.001) were noticed in oocyte and follicular diameters beyond 16 WOA. Other ovarian follicular morphometric traits showed inconsistent increases until 16 WOA. The first phase of yolk deposition occurred between hatching and 20 WOA, the second phase between 20 and 26 WOA, and the third and final phase between 27 and 28 WOA. Peripheral 17β-oestradiol concentrations increased from 20 WOA until it peaked at 28 WOA, while peripheral progesterone concentrations fluctuated considerably during sexual development. Both oestradiol and progesterone concentrations were negatively and moderately correlated (p<0.01) with oocyte nuclei diameter, granulosa and theca layer heights. However, oestradiol concentrations were positively and strongly correlated (p<0.001) with oocyte and follicular diameters, and moderately with progesterone concentrations. Three phases of yolk depositions were found in the guinea hen, with the final phase terminating at 28 WOA, at a much smaller oocyte diameter than in the domestic chicken. Oestradiol might play a role in yolk deposition in this species.

Comparison of Ovarian Morphology and Follicular Disturbances between Two Inbred Strains of Cotton Rats (Sigmodon hispidus)

Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.

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

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.

Stress-Induced Morphological Changes of Ovarian Histology in Female Wistar Rats

Stress as it relatesto infertility has become a global issue attracting public health concern. The present study examined the morphological changes in ovarian histology in response to oxidative stress-induced in female rats following exposure to different stressors. 92 rats of 12-14weeks old weighing between 120-160g were used for the study. Three (3) different stress models were utilized for stress induction at the rate of 1, 3, and 5hours per day for 1, 2, and 3weeks respectively. At the end of stress induction durations, bodyweights were obtained and the rats were euthanized via cervical dislocation while the ovary weights were carefully isolated and their weights recorded. The harvested ovary was sectioned, mounted on slides, stained, and observed under the microscope for histopathological investigations. Findings from this study established that exposure to restraint mirror or intruder stressor significantly (p<0.05) altered the body and/or ovary weights of the rats irrespective of the rate of exposure when compared to the control group. Cellular degeneration, infiltration, and atretic follicular changes were observed in the ovarian histology of rats in response to stress-inducedchanges caused by exposure to restraint or intruder stressors, whereas, equivalent exposure of the rats to mirror stressor did not result in any observed degenerative changes in the histology of the ovary studied. Our study revealed that exposure to restraint or intruder stressor points towards the existence of stress contributes towardsorgan/body weight changes and cellular damage inthe ovarian tissuespossiblycausing pathogenesis in reproductive capacity of females.

OR27-03 Duration of Testosterone-Induced Acyclicity Influences Corpora Lutea Formation and Stromal Changes in a Transgender Mouse Model

The impact and reversibility of long-term gender-affirming testosterone (T) therapy on the reproductive axis of transgender men has not been well-established. Little is known about outcomes for transgender men interested in pausing T therapy to harvest oocytes or get pregnant. We previously established a translational mouse model to investigate T-induced acyclicity and ovarian perturbations. We hypothesized that the duration of T-induced acyclicity would impact the reversibility of cyclic and ovarian changes. To test this hypothesis, T-treated mice were assigned to two groups: (SHORT) 6 weeks of T therapy with immediate reversibility (1.5 mg T propionate pellet implant/removal, n = 5) and (LONG) 6 weeks of T therapy with a prolonged T washout phase (subcutaneous oil injections of T enanthate at 0.9 mg once weekly, n = 5). Control groups (placebo pellets n = 5, sesame oil vehicle injections n = 5) were run in parallel. Estrous cycles were monitored using daily vaginal cytology. Following cessation of T therapy, mice were sacrificed in diestrus after resumption of cyclicity for 4 cycles and ovarian histology examined. Data were analyzed in GraphPad Prism using Welch’s t-test or Mann-Whitney where appropriate. T therapy led to persistent diestrus within a week after T administration for all T-treated mice and none of the controls. The total duration of acyclicity was 6±1 weeks for the SHORT group, which was significantly shorter than the 11±2 weeks for the LONG group (mean ± s.d., p = 0.0079). With resumption of cyclicity, both the SHORT and LONG groups had a significantly lower percentage of days in estrus and higher percentage of days in metestrus as compared to their parallel age-matched controls. Ovarian histology for the SHORT group all showed regular corpora lutea and minimal stromal changes, however, 3/5 mice in the LONG group lacked corpora lutea and 4/5 revealed marked stromal cell hypertrophy. Similar stromal cell changes were not seen in control mice. In conclusion, the length of time of T-induced acyclicity appears to impact the development of stromal cell hypertrophy and formation of corpora lutea even after resumption of cyclicity with similar alterations to the estrous cycles. These findings may have clinical relevance for transgender men interested in fertility, based on duration of gender-affirming T therapy. Future work will aim to separate out the respective contributions of T exposure and acyclicity to the stromal phenotype.