International Journal of

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Background: Many attempts have done to improve cryopreservation of mammalian ovaries using simple, economical and efficient technique “vitrification”.
Objective: The aim of the present study was to compare the mouse ovaries cryopreservation by direct cover vitrification (DCV) using different concentrations of ethylene glycol (EG) with conventional vitrification methods (CV).
Materials and Methods: Ninety NMRI mice were sacrificed by cervical dislocation; their ovaries were divided into three main experimental groups: control or non-vitrified group, CV group and DCV groups with 4, 6 and 8M EG as cryoprotectant. After vitrification-warming, the viability of mechanically isolated follicles and the morphology of ovarian follicles by light and electron microscopes were studied.
Results: The normality of primary and preantral follicles in non-vitrified and CV groups were higher than those achieved by DCV groups (p<0.001). The survival rates of isolated follicles in non-vitrified, CV and DCV groups with 4M, 6M and 8M ethylene glycol were 98.32, 96.26, 84.10, 85.46 and 84.56 %, respectively and in DCV groups it was lower than other groups (p<0.001). The ultrastructure of ovarian follicles was well preserved in CV technique. The follicles in DCV groups appeared to have vacuolated oocyte with nuclear shrinkage and irregular distribution of cytoplasmic organelles. Their mitochondria were located mainly in the sub cortical part of the oocyte and the granulosa cells demonstrated some signs of degeneration.
Conclusion: DCV of mouse ovarian tissue using only EG has induced some alteration on the fine structure of follicles. The integrity of mouse ovarian tissue was affected by DCV technique more than CV.

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Background: Oxidative stress is the most frequent cause of female infertility disorders including polycystic ovary syndrome (PCOS). Genistein as a major component of soybean isoflavone scavenges free radicals by antioxidant activities.
Objective: The present study examines the antioxidant effects of genistein on ovarian tissue following experimental PCOS in rats.
Materials and Methods: Twenty female Wistar rat were randomly divided into the following groups (n=5 each group): (I) control group (no treatment); (II) induced PCOS (injection of estradiol valerate); (III) genistein-treated non-PCOS (received genistein); and (IV) genistein-treated PCOS groups. The weight of rats were measured and the blood samples collected and centrifuged. The oxidant and antioxidant activity of plasma and ovaries were measured. All rats were sacrificed under anesthesia, and ovaries were collected and weighted. Histological examination and follicular quality
were assessed by staining.
Results: In histological observation, the induced PCOS rats displayed more number of atretic follicles and the follicular quality in genistein-treated rats was similar to the control groups. The plasma and ovaries malondialdehyde levels significantly increased in PCOS rats (p < 0.001), while the total antioxidant capacity levels, glutathione peroxidase, and superoxide dismutase activities significantly decreased (p < 0.001). The plasma and ovary  malondialdehyde levels significantly decreased in PCOS rats that were treated with genistein (p < 0.001) and the total antioxidant capacity (p < 0.05),
glutathione peroxidase, and superoxide dismutase activities significantly increased (p < 0.001).
Conclusion: Treatment with genistein preserved follicular quality by increasing antioxidant activities and scavenging oxidant levels in PCOS rats.

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Anti-Müllerian hormone (AMH) is made by the granulosa cells of preantral and small antral follicles which blocks the transition from the primordial to the primary follicular stage. Metformin may be associated with a decrease in AMH serum level and antral follicles in women who suffer from polycystic ovary syndrome (PCOs)
It was reported that metformin in women with polycystic ovary syndrome (PCOs) is associated with a decrease in both AMH serum level and antral follicles.
The objective of the current letter was the evaluation of the effects of metformin on hormonal profile of women with PCOs. In this prospective, randomized, doubleblind controlled clinical trial, 42 women (aged 17–45 yr) with PCOs who were randomly allocated to receive 500 mg Metformin orally three times a day or placebo for three months were included. Fasting plasma glucose, follicular stimulating hormone, luteinizing hormone (LH), prolactin (PRL), testosterone and AMH levels were measured at baseline and at the end of the period. In this study, independent and paired t-test were used for quantitative comparison and chi-square analysis for qualitative variables. Logistic regression analysis was done to identify independent risk factors, and Pvalue of lower than 0.05 were considered significant. All of the statistical analyses were done by SPSS software, version 20.0. We didn’t find any significant change after the study between treatment and control groups in hormonal profiles especially AMH. However, in subgroup analysis, we revealed that AMH and LH levels decreased significantly in normal weight patients (p= 0.024, 0.048, respectively) and prolactin levels in subgroup of overweight patients (p= 0.001). Moreover, patients in metformin group at the end of study had more regular menses, more weight loss, and lower hair loss (p= .001, 0.04, 0.014, respectively). Women with PCOs have elevated levels of LH that is secondary to increased sensitivity of pituitary to GnRH. Increased levels
of LH lead to hyperandrogenism. Metformin can improve this condition. In this study, we observed that LH was decreased significantly in the metformin group of PCOs patients (p= 0.05). It is in agreement with some of the previous studies, however, De Leo and et al. reported a non-significant decrease in LH levels after treatment with metformin (1). Pieces of evidence showed that AMH levels in PCOs patients are 2 to 3 times higher than age-matched normal women (2), and this condition is a marker for PCOs and can be a surrogate test of hyperandrogenism (3). Metformin in our study improved the irregularity in the menstrual cycle (p< 0.001), hair loss (p= 0.014), and prolactin secretion (p= 0.024), as well as LH secretion (p= 0.05); however, it didn’t show any significant decrease on AMH levels. Metformin can reduce ovarian volume in PCOs patients especially in hyperinsulinemic subgroups (4); however, we observed that in normal weight subgroup of patients, metformin decreased AMH significantly (p= 0.024), and in overweight or obese patients, prolactin decreased in response to metformin, significantly (p= 0.001). Moreover, metformin leads to improvement in follicular development with fewer percentage of preantral follicles and cysts and higher percentages of antral follicles (5). Considering these pieces of evidence, it seems that AMH after metformin treatment in PCOs patients was secret in comparable amounts of pretreatment; however, the source of secretion after starting metformin is different from pretreatment and switched from small follicles and cysts to more developed follicles and structures such as antral follicles and corpora lutea. Further investigations that can differentiate secreted AMH from a different source will shed more light on this field. The most important limitation of our study is the small sample size. GI side effects of metformin caused some patients in metformin group to refuse getting the treatment. Studies with more attendants can display the difference between different phenotypes of PCOs patients in response to metformin. In conclusion, this study showed that
metformin can cause favorable effects on the hormonal profile of PCOs patients, and although AMH levels were not decreased significantly, menstruation improved in the treated group.
The most important limitation of our study is the small sample size. GI side effects of metformin caused some patients in metformin group to refuse getting the treatment. Studies with more attendants can display the difference between different phenotypes of PCOs patients in response to metformin. In conclusion, this study showed that metformin can cause favorable effects on the hormonal profile of PCOs patients, and although AMH levels were not decreased significantly, menstruation improved in the treated group.
 

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