Protective effects of licorice extract on ovarian morphology, oocyte maturation, and embryo development in PCOS-induced mice: An experimental study

Abstract Background Polycystic ovary syndrome (PCOS) is an oxidative state resulting in ovarian dysfunction. Licorice is one of the natural antioxidants used for the treatment of infertility. Objective To evaluate the effect of licorice extract on ovarian morphology, oocyte maturation, and embryo development in PCOS-induced mice. Materials and Methods thirty-two female NMIR mice were divided into four groups (n = 8/each): control group receiving no treatment (group I); PCOS group injected with estradiol valerate once daily for 21 days (group II); and experimental groups receiving either 100 mg/kg (group III) or 150 mg/kg (group IV) licorice by gavage along with estradiol valerate once daily for 3 wk. Serum levels of the testosterone and estrogen were measured using ELISA kit. Histological study of ovaries was evaluated, and oocyte maturation, fertilization rate, and embryo development were determined after in vitro maturation. Results Experimental groups (III, IV) had significantly higher testosterone and estradiol levels compared to the PCOS group (p ≤ 0.001). A significant increase in the number of healthy follicles (primary, preantral follicles) (p = 0.001), corpus luteum (p = 0.001) with significant decrease in the number of atretic follicles (primary, preantral, cystic follicles) (p ≤ 0.001) was seen in the experimental groups. Increase in the fertilization rate (p ≤ 0.001) and blastocyst stage embryos (p = 0.02, p = 0.004) were observed in the experimental groups. Conclusion It appears that the two doses (100 mg and 150 mg) of licorice could decrease ovarian cyst and improve the fertilization rate of oocyte and embryo development in PCOS mice. However, there was no statistically significant difference between the two experimental groups.


Introduction
. In addition, the biosynthesis of estrogens from androgens is catalyzed by aromatase; hence, a deficiency in aromatase activity may lead to intraovarian disturbances and hormonal imbalance (5).
An ovulatory cycle may lead to infertility resulting in a need for assisted reproductive techniques (6). Moreover, in vitro maturation (IVM) is a relatively new method in assisted reproductive techniques, in which immature oocytes are extracted from the ovaries without gonadotropin stimulation, thus limiting the risk of ovarian hyperstimulation syndrome compared to in vitro fertilization (IVF) technique (7). Currently, using various herbal remedies as natural antioxidants is suggested for improving all clinical features of PCOS to recover the menstrual cycle and normal serum hormones levels. Therefore, antioxidant supplementation are known to be effective in reducing testosterone, polycystic ovaries, and improving reproductive cycles (8,9).
licorice (Glycyrrhiza glabra) has been used to treat different diseases for many years (10).
Multiple biological activities such as the powerful antioxidative, antifatigue, antibacterial, antiviral, antiproliferative, and estrogenic activity of licorice have been demonstrated in several experimental evidence (11,12). Therefore, some studies have suggested licorice extract as a possible candidate for the treatment of infertility as well as PCOS (13).
Previous studies have revealed that blocking the activity of 3β-hydroxysteroid dehydrogenase (3HSD), 17-hydroxysteroid dehydrogenase (17HSD), and 17-20 lyase, and stimulating the activity of aromatase, while also affecting the 5a-and 5b-reductase activity, are some effects of licorice extract (14,15). All these enzymes are involved in the synthesis and/or metabolism of androgens and estrogens (14). Lakshmi

Chemicals
Material and reagents used in the present study were purchased from Sigma-Aldrich (Germany), unless otherwise mentioned.

Animals and experimental design
Adult Female (6-7 wk old) and male (8-12 wk old) Naval Medical Research Institute (NMRI) mice were obtained from the breeding center at the Faculty of Science, Urmia University (Urmia, Iran).
The mice were housed in a 12-hr light/dark cycle, at 25 ± 1°C and 50-60% humidity, and had free access to standard pellet diet and water ad libitum.
The female mice were randomly assigned into four groups (n = 8/group) as follows: • Group I: mice receiving no treatment (control group) • Group II: PCOS was induced in mice through 0.2 mg estradiol valerate intramuscularly once daily for 3 wk.

Preparation of licorice extract
The licorice was collected from a local farm in

Blood sampling
After the injection, the female mice were sacrificed under anesthesia, before drawing blood (2 mL) from the heart. Blood serum was separated by centrifuging the blood samples at 6000 g for 5 min. Serum samples were stored at -70°C for future analysis. Serum testosterone and estrogen concentrations were assessed through immune-radiometric techniques using commercially available Elisa kits (diaplus, USA) according to the manufacturer's guidelines (23).

International Journal of Reproductive BioMedicine
Shamsi et al.

Histological analysis
Ovarian tissue was removed for histological examination following fixation in 10% formalin.
Afterward, the samples were cut serially at

Oocyte collection
The

In vitro maturation (IVM)
The IVM medium consisting of complete

IVF
The oocytes in the MII stage were obtained from the different groups and fertilized by sperm

Ethical consideration
All animal experiments and study protocols were approved by the educational assistant of the Faculty of Science, University of Urmia, Urmia, Iran.

Statistical analysis
Data presented as mean ± standard deviation (SD). The differences between groups were analyzed by ANOVA, followed by Tukey's test using the SPSS software (Statistical Package for the Social Sciences, version 19. P ≤ 0.05 was deemed statistically significant.

Hormonal concentrations
As shown in Table I

Ovarian morphology
In the control group, ovarian tissues had normal histological appearance (Figure 2a)  (Table II). Morphological studies in the PCOS group showed that the number of atretic follicles PRIF (p = 0.01), PF (p = 0.002) in this group were significantly higher compared to the control group (Table III). Also, an evaluation of the number of atretic follicles PF (p ≤ 0.001), PAF (p = 0.02, p = 0.03) showed a significant decrease in the experimental groups compared to the PCOS group (Table III).
The PCOS group showed a highly significant increase in CF (p = 0.001) (Figure 2b).  (Table   IV).

IVM of oocytes
As shown in Table V, (Table V).

Group IV
1.60 ± 0.14 * * 10.05 ± 0.35 * * Data presented as Mean ± SD. one-way ANOVA test, *, ** Significant difference between the PCOS and other groups at p < 0.05, and p < 0.01, level respectively Significant difference compared to the control group Data presented as Mean ± SD. one-way ANOVA test, *, **, *** Significant difference between the PCOS and other groups at p < 0.05, p < 0.01, and p < 0.001 level respectively; Significant difference compared to the control group; Significant difference compared to the PCOS group Data presented as Mean ± SD. one-way ANOVA test, * , **, *** Significant difference between the PCOS and other groups at p < 0.05, p < 0.01, and p < 0.001 level respectively. Significant difference compared to the control group; Significant difference compared to the PCOS group International Journal of Reproductive BioMedicine Shamsi et al.

Discussion
In the present study, the PCOS phenotype was They also studied the beneficial effects of licorice extract on oocyte maturation and infertility due to its phytoestrogen properties (38).

Conclusion
According to the results of the present study, both doses of licorice may act as a useful treatment for improving PCOS. Our findings support that oral licorice extract intake can cause a reduction in testosterone and estrogen levels, and significantly enhance oocyte maturation, fertilization, and embryo developmental rates in mice. Also, licorice extract can improve ovary morphology in PCOS-induced mice. However, there was no statistically significant difference between the two experimental groups. Since no difference was found between the doses of 100 mg and 150 mg, these conditions will determine the final dose of atorvastatin for each mouse.